CN1303394A

CN1303394A - Peptide compound for preventing and treating HIV infection and immunity diseases

Info

Publication number: CN1303394A
Application number: CN99806643A
Authority: CN
Inventors: 王长怡
Original assignee: United Biomedical Inc
Current assignee: United Biomedical Inc
Priority date: 1998-06-20
Filing date: 1999-06-21
Publication date: 2001-07-11
Anticipated expiration: 2019-06-21
Also published as: CA2330235A1; CN1207306C; HK1036805A1; TWI227242B; BRPI9912175B8; EP1098910B1; JP4216473B2; WO1999067294A1; ES2336393T3; EP1098910A1; JP2002518523A; CA2330235C; BRPI9912175B1; ATE447585T1; AU766955B2; AU4704899A; DK1098910T3; EP1098910A4; ZA200006385B; US6090388A

Abstract

The invention provides peptides comprising a sequence homologous to a portion of the CDR-2 like domain of CD4, covalently linked to a helper T cell epitope, and optionally to other immunostimulatory sequences as well. The invention provides for the use of such peptides as immunogens to elicit the production in mammals of high titer polyclonal auto-antibodies, which are specific to CD4 surface complex. These auto-antibodies prevent binding of HIV viral particles to CD4+ cells. The peptides are useful in pharmaceutical compositions, to provide an immunotherapy for HIV infection and to protect against HIV infection.

Description

Be used to prevent and treat the peptide combinations of HIV infection and Immunological diseases

Invention field

The present invention relates to peptide combinations as immunogenic application, each peptide that wherein comprises comprises a target antigen site by the antibody recognition of anti-host cell HIV acceptor/coreceptor mixture.This mixture comprises the CD4 relevant with chemokine receptor domain.The target antigen site is linear covalently bound ring-type, its be connected in series (1) by a kind of carrier proteins of chemical coupling, or preferably (2) by chemical coupling or more preferably by direct synthetic a kind of peptide helper T cell epitope and other immunostimulation peptide sequence of connecting.More particularly, the present invention relates to this peptide combinations as inducing healthy Mammals to comprise that the people produces the immunogenic application of high-titer antibody, described antibody has former generation strain isolated and the neutralization activity widely of 2 type HIV (HIV-2) strain isolateds of former generation of anti-1 all clade of type HIV (HIV-1).The invention still further relates to described peptide combinations as immunogen prevention with treat immunodeficiency virus infection and treat for example for example rheumatoid arthritis, systemic lupus erythematous and psoriasic method of transplant rejection, autoimmune disorders of undesirable immunne response.

Background of invention

Though because the discovery of HIV and characterizing, also there has been major obstacles in the development of HIV vaccine and immunotherapy since 14 years to the existing further investigation of its vaccine.These obstacles comprise the variability of HIV-1, this virus structure is lacked understanding and prevention HIV is infected essential immunne response be short in understanding.Referring to D.Burton and J.Moore, natural drug (NatureMedicine), 1998,4:496-48.The head of the AIDS vaccine research council of United States Government claimed on February 1st, 1998, the safe vaccine of prevention AIDS still needs more than 10 years apart from test, much also is in unknown state (http: ∥ cnn.com/HEALTH/9802/01/aids.vaccine.search) because how to work still to have about the health immunity system.

Previously (for example for effective Recombinant HIV-1 coating subunit vaccine, gp120 and gp160 vaccine product) very optimistic, known vaccine serum from several clinical experiments can be external in and the laboratory chorista (Belshe etc. of HIV-1, U.S. psoriasis state's AMA magazine (J.Am.Med.Assoc.), 1994,272:475; Keefer etc., AIDS research, human reverse transcript virus (AIDS Res.Hum Retroviruses), 1994,10:1713).When find this vaccine serum in and the former generation patient of HIV-1 chorista major part invalid (Hanson, AIDS research, human retrovirus, 1994,10:645; Mascola etc., the transmissible disease magazine, 1996, in the time of 173:340), this optimism has shaken.These disappointed results cause NIH to postpone the huge extensive effect experiment of cost of several recombinant envelope proteins based on the HIV subunit vaccine in June, 1994 decision.

The research of HIV vaccine at present concentrates on former generation strain isolated, and it is considered to causing aspect the human infection than similar HIV strain system of normally used laboratory strains system (Sawyer etc., Journal of Virology, 1994,68:1342; Wrin etc., Journal of Virology, 1995,69:39).Former generation strain isolated of HIV-1 carries out restricted cultivation by patient PBMC or blood plasma and the PBMC that do not infect and obtains.Can by the phenotype that describes below easily with former generation virus from human T lymphocyte system, go down to posterity several times and in these T clones the T clone of good growth adapt to (TCLA) virus for example among III b/LAI, SF2 and the MN explanation come out:

(1) do not resemble TCLA virus, most of former generation strain isolated be difficult in T clone, growing.

(2) not resembling TCLA virus is that full synplasm is epigamic, former generation strain isolated be included in the pairing cell space that PBMC induces synplasm to form in cultivating and induce (SI) chorista and non-synplasm to induce (NSI) chorista.In SI strain isolated of former generation, great majority will, especially in the responsive T clone of HIV-MT2, duplicate, and seldom can for example duplicate among CEM or the H9 in the almost objectionable T clone that is generally used for cultivating the TCLA chorista.Non-synplasm induces (NSI) former generation strain isolated only to duplicate in former generation T cell.

(3) strain isolated has the height resistance in the neutralization of external reorganization soluble form to virus receptor PROTEIN C D4 (rsCD4) former generation, rsCD4 (the Daar etc. that need many times of 200-2700 for equivalent neutralization ratio TCLA strain system, PNAS USA, 1990,87:6574-6578).

(4) strain isolated is also used the vaccine-induced antibody of gp120 (coating) to neutralization and is had resistance former generation.Comparatively speaking, TCLA strain system is sensitive (Sawyer etc., Journal of Virology, 1994,68:1342 for the neutralization that peplos is had specific antibody; Mascola etc., 1996).

In former generation,, these phenotypic characteristics of strain isolated resulted from the still unconversant constitutional features of HIV, especially with the characteristic that is difficult to study of anti--peplos that env antibody is relevant (D.Burton and J.Moore, natural drug, 1998,4:495-498).The variability of virus, a kind of yielding characteristics also becomes an obstacle (Mascola etc., 1996) of developing the effective HIV vaccine of worldwide.These factors cause the failure of not anticipating at the AIDS vaccine of virus of the anti-TCLA homotype strain system that is easy to grow of development jointly.A kind of alternative route of the HIV of development vaccine can be carried out by the following method, disturbs the HIV acceptor of host cell, blocks infection by preventing that HIV from combining with permissive cell or merging thus.Research at cell provides HIV coating height variability and the multifarious method of phenotype of overcoming.

The research at cell that prevents that HIV from infecting has been pointed out in the research of active in the SIV rhesus monkey macaque model and passive immunization, its show anti--cell antibody be devoted to effectively to be protected from infections (Stott, natural, 1991,353:393).In addition, the monoclonal antibody of anti-CD4, a kind of TXi Baoshouti of MHC class and HIV bonded primary receptor, known for a long time its in the HIV-1 neutralization analysis with the CD4 epi-position but not virus strain's dependency mode is blocked infection (Sattentau etc., science, 1986; 234:1120).In this immunoprophylaxis research, have been found that anti--CD4 monoclonal antibody block effectively cell by former generation strain isolated infect (Daar etc., NAS's scientific advance, 1990,87:6574; Hasunuma etc., Journal of Immunology, 1992,148:1841).

Other potential research that effectively relates to cell comprises target Chemokine Receptors CXCR4, CCR5, CCR2b and CCR3, and they are accredited as coreceptor (Feng etc., science, 1996, the 272:872 of HIV recently; Doranz etc., cell, 1996,85:1149).These coreceptors are with the CD4 functionating, and the later stage binding interactions of starting viral envelope glycoprotein and host cell membrane and the later stage that retrovirus duplicates are invaded step (Chackerian etc., Journal of Virology, 1997; 71:3932).

Carry out effective HIV combination and merge needing CD4 and coreceptor to show that these molecules one or both of all may be that cell-guiding strategy suppresses the fine target of infection.The antibody of anti-host cell CD4/ coreceptor mixture shown influence combination that HIV infects and later stage integrating step (Wang, WO97/46697).Induce (SI) and non-synplasm to induce the virus of (NSI) strain system to transmit to cell with the synplasm of HIV in these antibody to cell or cell.

In conjunction with the chemokine antagonists of CCR5 also show can prevent effectively SI and NSI virus infection (Simmons etc., science, 1997,276:276).In and the NSI chorista especially meaningful because it is believed that NSI strain system is responsible for most of HIV and transmits, and often in and the anti-HIV antibody of TCLA chorista have resistance (Fauci, 1996).The reagent of target HIV cell receptor make people not demand side and provide neutralization activity (Chen etc., Journal of Virology, 1997, the 71:2705 of potential in addition to peplos variation phenotype and high variability at HIV-2 and SIV; Pleskoff etc., Journal of Virology, 1997,71:3259; WO97/46697).

According to the patent application (WO97/46697) of a common pending trial, promote viral combination and invade host T cell, comprise that the CD4 on the host T cell surface and the host cell receptor/coreceptor mixture of chemokine coreceptor are the effective targets of neutralizing antibody.In this application, the contriver has proved that resulting antibodies specific ground resists the antigenic compound of this cell surface.In the cell-surface antigens answering on former generation strain isolated neutral HPB-ALL cell, do not cultivate other anti--cell antibody by HIV-1.The antibody of describing in this application with required character can be blocked monkey and be infected by HIV-2 in Infection in Vitro and blocking-up people cell by diversified phenotype and genotypic HIV-1 strain isolated of former generation by infection in the HIV-1 body, people's cell by the human immune system that SIV infects, rebuilds in mouse.The target of these cell-surface antigens mixtures that comprise the associating of CD4 acceptor and chemokine coreceptor become protectiveness anti--cell antibody.

Anti-CD4/ is common-anti--cell antibody of receptor complex than anti--antiviral antibody of antiviral coating show more effective decorrelation HIV strain system in and pattern.Shown in the application (WO97/46697) of common pending trial, the monoclonal antibody of the anti-HPB-ALL of generation (MAb B4) has middle isoreactivity to solubility CD4 (rsCD4) protein of reorganization, simultaneously strongly in conjunction with the HPB-ALL cell.Discovery to the specificity of CD4/ coreceptor cell surface mixture in and HIV-1 strain isolated camber of former generation effective, but in and nearly unavailable in the TCLA strain system.Comparatively speaking, anti--env antibody is to the opposite pattern of preferential neutralization performance of TCLA strain system.

Find MAb B4 in external microplate is analyzed with the concentration of＜10 μ g/ml in and HIV strain isolated of former generation.Comparatively speaking, for the solubility CD4 (rsCD4) of reorganization have high titre (＞5Log10) specific polyclonal antibody not performance to any neutralization activity of HIV strain isolated of former generation, although they have intensive T cell in conjunction with activity.Therefore, former generation strain isolated possibility pair cell surface C D4/ coreceptor antigenic compound has specific antibody comparison and has preferentially sensitivity of the specific antibody of simple CD4.The HIV neutral that is undertaken by the antibody of anti--CD4/ coreceptor mixture extensively characterizes and comprises MAb B4 and its analogue MAb M2 and MAb B13 (WO97/46697).

At CD4/ common-the active mechanism of neutralization widely of the antibody of receptor complex is not clear, because the effect in mediation HIV course of infection of those cell surface mixtures is changeable, as have with those antibody shown in the ability that influences combination that HIV infects and later stage integrating step (Wang, WO97/46697).CD4/ coreceptor cell surface mixture may have dual function in mediation HIV infection and pathogenic process: the T cell surface receptor that (1) is merged by HIV and invaded as HIV combination, cell; Or (2) are as the HIV supressor.

Yet though these materials infect effectively suppressing HIV, the antagonist or the antibody of above-mentioned cell-sensing can not be used as preventative vaccine.The cell of Miao Shuing-sensing antagonist or antibody (WO97/46697) were not immunogens in the past, and can not be used as preventative vaccine.They are passive immunizing agents.Need frequently use these materials, to keep their effect of serum-concentration competence exertion that whole acceptors are occupied.By active immunity induce the active of anti-CD4/ coreceptor mixture anti--vaccine that autoantibody is replied should be preferred for protective immunity.This vaccine if can be developed out, should provide permanently effective anti-infective protection, needs a small amount of immunogen frequently and conveniently not use simultaneously.

For effect, the immunogen composition of this class vaccine must comprise or a similar B cell epitope, related locus on host cell receptor/coreceptor mixture, have the reliability that is enough to induce intersection-inhibition antibody, keep the locus specificity that is enough to avoid unfavorable immunosuppressive action simultaneously.Though in and anti--cell antibody of HIV, comprise that having the active anti-CD 4 antibodies of neutralization can obtain, the simulation site of this class synthetic antigen also is not easy to identify at present.The neutral of having reported that is used for resists-CD4 monoclonal antibody (Burkly etc., Journal of Immunology, 1992,149:1779) and the extensive neutral of report such as Wang anti--the discontinuous configuration site of not transreplication on monoclonal antibody (WO97/46697) the identification CD4 of CD4/ coreceptor.The accurate knowledge that lacks vulnerable site is selected useful host cell antigenicity target and it is duplicated very difficulty from long recombinant immune is former.The most of antibody deficiencies that obtain by the CD4 immunity have practical value specificity (Davis etc., nature, 1992,358:76).For example, during the high immune antiserum(antisera) of the high titre of rsCD4 lacks and the activity (WO97/46697) of HIV strain isolated of former generation.And, to the extensive region of T cell antigen have the excessively immunosuppression of extensive reactive antibody (Reimann etc., AIDS research, the human retrovirus, 1997,13:933).

In addition, though a large amount of drawing of CD4 research produced molecule the structure function collection of illustrative plates (Sattentau etc., science, 1986,234:1120); Peterson and Seed, cell, 1988,54:65; Jameson etc., science, 1988,240:1335; Sattentau etc., The Journal of Experimental Medicine, 1989,170:1319; Hasunuma etc., Journal of Immunology, 1992,148:1841; Burkly etc., Journal of Immunology, 1992,149:1779; Davis etc., nature, 1992,358:76), these collection of illustrative plates do not provide the enough accurate structural models in prediction susceptible effector site, are synthetic peptide thereby it may be duplicated into.Disclosed CD4 model does not disclose the immunogen based on CD4 of usefulness.

Except suitable site-specificity, effectively acceptor/coreceptor the immunogen of HIV vaccine must have the hyperimmunization hormesis, so that induce the antibody response of enough generation protection levels.These immunogens also must be designed to be able to overcome the strong tolerance to self molecule performance.Therefore, also need to possess the immunogen of enough immunizing power.

One object of the present invention is to provide has the required locus specificity and the peptide combinations of immunizing power, as the immunogen of prevention HIV infection.

The improved immunogenicity of useful synthetic peptide based immunogens of the present invention and suitable the specificity combination of the method by a series of evaluations and design synthetic peptide based immunogens realize.These methods comprise: (1) a kind of program of identifying effective high affinity target epi-position effectively; (2) stablize the method for the constitutional features of the target site on the synthetic peptide by introducing annular restriction (cyclic constraints), thereby make cross reaction maximization self molecule; (3) by with it with contain extensively reactive t helper cell (Th) epi-position that mixes and mix the immunogenic method of enhancing B cell target epi-position; (4) chemistry of peptides by application combination increases the method for the repertoire of t cell epitope, further adapts to the immunologic responsiveness of the variation of outbred population thus.

Synthetic peptide is used to " epitope mapping ", is used to develop the immune structure territory determinant or the epi-position of the protein surface of novel vaccine and diagnostic reagent with evaluation.Epitope mapping is used the overlapping peptide of corresponding zone on a series of target proteins, participates in the interactional site of antibody-immunogenic determinant to identify.Usually, the peptide that the epitope mapping application length is relatively short is with the linear determinant of accurate detection.The fastest known epitope mapping method is PEPSCAN, and it is based on synthetic hundreds of simultaneously the length that are coupled on the solid support is 8-14 amino acid whose overlapping peptide.Detect the ability of the binding antibody of coupling peptide.PEPSCAN research is effective aspect the LINEAR CONTINUOUS determinant of location, but for identifying that the required epi-position in the discontinuous effector of simulation site is invalid, for example HIV acceptor/common-receptor binding site (Meloen etc., Ann Biol.Clin., 1991,49:231-242).A kind of alternative method depends on the nested overlapping peptide synthetics that a series of length ranges do not wait from 15-60 residue.These long peptides are reliably, but need synthesize by a series of independently solid phase methods of peptide synthesis loaded down with trivial detailsly, rather than synthetic by quick and synchronous PEPSCAN.The a series of nested overlapping peptide that obtains can be used for analyzing antibodies at for example experiment immunization and neutralization infection system then, so that evaluation comprises the immune structure territory determinant that the best of simple discontinuous epi-position is presented.This method by Wang etc. about site, immune structure territory mapping research example explanation from HTLV I/II (US5476765) and HCV (US 5106726).It is used to select the best exact position that occurs of HIV neutralizing epitope on the gp120 sequence (Wang etc., science, 1991,254:285-288).

Peptide based immunogens is more pliable and tougher than protein usually, is difficult for keeping any preferred structure.Therefore to come the stabilized peptide immunogen be useful by introducing the annular restriction.The conformation of target epi-position can be simulated and keep to a kind of peptide based immunogens of accurate cyclisation, induces the antibody that has cross reactivity on the real molecule with this site thus.For example, by adding the cysteine residues of favourable arrangement, then through the sulfydryl cyclisation, can on synthetic peptide, duplicate the ring structure (Moore that is present on the real epi-position more accurately, synthetic peptide chapter 2: users' guidebook, ed.Grant, WH Freeman andCompany:New York, 1992, pp/63-67).

Immunogenic another important factor that influence derives from the peptide based immunogens of acceptor/coreceptor mixture is by the t helper cell epi-position with host T-helper acceptor and class reaction this peptide to be to pass immunity system (Babbitt etc., nature, 1985,317:359-361).T-assists epi-position (Th) to be provided by carrier protein usually, because being difficult to produce misrouting and the carrier inductive epi-position that the peptide-carrier binding substances of accurate qualification, most of antibody reply carrier suppresses to make described carrier protein have shortcoming (Cease simultaneously, InternRev Immunol, 1990,7:85-107; Schutze etc., Journal of Immunology, 1985,135:2319-2322).Perhaps, the T cell is auxiliary can stimulate by the synthetic peptide that contains the Th site.Therefore, be called as the II class Th epi-position that mixes Th and induce effective T cell auxiliary, and can the synthetic B cell epitope of reduced immunogenicity mixes with self possessing very, to produce effective peptide based immunogens (US 5759551).Design good mix Th/B cell epitope chimeric peptide can induce Th to reply and most of members of the haploid kind diversity of consequent target performance diversity MHC population in the antibody response in B cell site.Mixing Th can provide by the specific sequence that derives from effective exogenous antigen, for example Measles virus F albumen, hepatitis B virus surface antigen and chlamydia trachomatis major outer membrane albumen (MOMP).Some known Th of mixing have been presented at that to strengthen the reduced immunogenicity peptide aspect be equivalent to the decapeptide hormone be effectively (US 5759551).

Mixing Th epi-position length range is about 40 amino-acid residues of about 15-(US5759551), has the common constitutional features usually, can contain the specificity marker sequence.For example, common trait is the amphipathic molecule spiral, and it is that hydrophobic amino acid residue occupies spiral one side, electrically charged and polar residues occupy on every side the α-Luo Xuanjiegou of face (Cease etc., NAS's scientific advance, 1987,84:4249-4253).Epi-position contains other one-level aminoacid pattern usually, and for example Gly or charged residue heel are followed an electrically charged or polar residues thereafter successively with two to three hydrophobic residues.This pattern description a kind of pattern that is called as the Rothbard sequence.In addition, epi-position is followed 1,4,5,8 principles usually, one of them with the residue of positive electric charge after the 4th, the 5th and the 8th follow hydrophobic residue, consistent with 1,4,5,8 both sexes spiral that is positioned on the same side.Hydrophobic, electrically charged and polare Aminosaeren constitutes because all these structures are by common, so each structure can exist (Partidos etc., hereditary Journal of Virology (J Gen Virol), 1991,72:1293-99 simultaneously in same Th epi-position; Alexander etc., immunology, 1994,1:751-761).Even be not all,, great majority contain at least one above-mentioned periodicity but mixing t cell epitope.These features can be inserted into the design in " idealized artificial T h site ".

The Th epi-position that mixes that derives from foreign pathogens for example comprises, but is not limited to hepatitis B surface antigen and cAg helper T cell epitope (HB _sTh and HB _cTh), Toxins, pertussis helper T cell epitope (PT Th), tetanus toxin helper T cell epitope (TT Th), Measles virus F albumen helper T cell epitope (MV _FTh), chlamydia trachomatis major outer membrane albumen helper T cell epitope (CT Th), diphtheria toxin helper T cell epitope (DT Th), plasmodium falciparum ring sporophyte helper T cell epitope (PF Th), schistosoma mansoni trisaccharide phosphoric acid salt isomerase helper T cell epitope (SM Th), and intestinal bacteria TraT helper T cell epitope (TraT Th).SEQ IDNOS:2-9 and the 42-52 of pathogenic agent deutero-Th in US 5759551 lists; The auxiliary site P11 of chlamydozoan is at Stagg etc., immunology, and 1993, list among the 79:1-9; HBc peptide 50-69 is at Ferrari etc., Journal of Clinical Investigation, and 1991, list among the 88:214-222.

Useful Th site can also comprise the Th of combination, and it inserts the degeneracy site of selecting in the design in Utopian Th site.In (WO 95/11998) such as Wang, a class makes up epi-position especially and is referred to as " structural synthetic antigen library " or SSAL.The Th of structure such as SSAL epi-position is made up of around a kind of position substituent of structural framing of constant residue tissue.The sequence of SSAL is determined by the one-level aminoacid sequence that arrangement mixes Th, keeps geostationary residue on the position that constitutes Th peptide unique texture, and provides degeneracy on the position relevant with identification polytropy MHC limiting element.MHC-is in conjunction with constant and variable position and preferred amino acid tabulation existing open (Meister etc., vaccine, 1995, the 13:581-591 of locus; Alexander etc., immunology, 1994,1:751-761).

All members of SSAL produce simultaneously with target B cell epitope and other sequence in a single solid-phase peptide is synthetic.Th library sequence has kept the structure gene seat that mixes Th and provides extensive haploid reactivity.For example, obtain from Measles virus F protein after the specific admixture epi-position, simulation is described to the degeneracy Th epi-position (Partidos etc., 1991) of SSAL1TH1.SSAL1TH1 and LHRH target peptide are together used.As the measles epi-position, SSAL1TH1 follows Rothbard sequence and 1,4,5,8 rules: 15 10 15Asp-Leu-Ser-Asp-Leu-Lys-Gly-Leu-Leu-Leu-His-Lys-Leu-As p-Gly-LeuGlu Ile Glu Ile Arg Ile Ile Ile Arg Ile Glu IleVal Val Val Val Val Val ValPhe Phe Phe Phe Phe Phe Phe

Charged residue Glu or Asp are added in position 1, to increase Th hydrophobic side electric charge on every side.At both sexes spiral hydrophobic side 2,5,8,9,10,13 and 16 keep hydrophobic residue then, and 2,5,8,9,10,13 and 16 mutability provides a kind of surface that has in conjunction with extensive MHC limiting element ability.The quiet effect of SSAL feature has been to increase the immunosuppressant scope (WO95/11998) of artificial T h.

According to the present invention, mix Th epi-position or the Utopian Th of mixing and Utopian SSAL Th epi-position and designed effective peptide based immunogens HIV with accurate epi-position collection of illustrative plates, ring-type restriction, insertion.This peptide is preferred, because they are safely and effectively.Owing to by accurate location and cyclic action and be widely used reactive Th and reply effectively presenting of HIV acceptor/coreceptor binding site that epi-position optimizes, believe that peptide based immunogens of the present invention provides immune efficacy.

Summary of the invention

Peptide combinations of the present invention comprises one or more peptide based immunogens as above-mentioned design.This peptide combinations is the basis that effectively prevents and treat the vaccine of HIV infection and Immunological diseases.Owing to peptide component of the present invention present in and the acceptor/coreceptor effector site in the CDR2-spline structure territory of CD4 become preferably.These peptides are because (1) has the locus specificity of optimization, its by CDR2-spline structure territory accurate epitope mapping and consider that the cyclic action that self configuration of CDR2 is selected obtains, and (2) their extensive reactive Th replys, and makes them induce effective antibody response.

According to the present invention, one or more peptides are provided, every kind of peptide comprises two peptide sequences in the sub-site of respective effects on the CDR2 structural domain that is positioned at CD4, or its immunologic function analogue.

In addition, by the covalently bound carrier protein of chemical coupling, or more preferably by the covalently bound synthetic immunostimulating composition of chemical coupling (for example mixing the Th epi-position), or more preferably by the synthetic stronger immunogenicity of target site of giving peptide of the present invention of direct peptide.The specific examples of carrier protein and immunostimulating composition is existing to be provided, for example, the BEDT-TTF A (PEA) of keyhole limpet hemocyanin carrier, modification, Th epi-position are (for example, SEQ ID NO:6) and general immunostimulatory peptides (for example, the invasion peptide (Inv) of Yersinia (SEQ ID NO:7).

Synthetic peptide of the present invention can be expressed from the next: (A) _n-(Th) _m-(B) _o-(CD4-CDR2 antigen peptide)-X

Or (A) _n-(CD4-CDR2 antigen peptide)-(B) _o-(Th) _m-X

Or (CD4-CDR2 antigen peptide)-(B) _o-(Th) _m-(A) _n-X

Or (Th) _m-(B) _o-(CD4-CDR2 antigen peptide)-(A) _n-X is wherein:

Each A is an amino acid independently, or a kind of general immunostimulatory peptides;

Each B is an amino acid or other chemical bond independently;

X is an amino acid whose α-COOH or α-CONH ₂

Th is a helper T cell epitope or an immunostimulant analogue or its fragment;

" CD4-CDR2 antigen peptide " is a kind of peptide antigen that can induce the antibody that reacts with the CD4 surface complex;

N is the number from 1-about 10;

M is from 1 to about 4 number; With

O is from 0 to about 10 number.

Peptide combinations of the present invention comprises from about 30 to about 115 amino-acid residues, preferably from about 40 to about 90 amino-acid residues, more preferably from about 50 peptide based immunogens to about 80 amino-acid residues.

Composition of the present invention randomly further contains adjuvant and/or drug administration carrier and other adds the conventional ingredient of vaccine preparation.The invention provides the dosage explanation, so that the immunotherapy antibody in the target CD4-CDR2 effector site that creates antagonism.

The present invention provides the synthetic peptide that can induce antibody in mammalian body first, and its protectiveness antagonism is infected from the HIV of multiple clade strain isolated of former generation.

When the host is used the vaccine preparation that contains peptide combinations of the present invention; the antibody response of peptide combinations of the present invention is provided the protection or the treatment of anti-HIV infection to the host by following several respects: (1) blocking-up HIV combines with the CD4-express cell; (2) HIV-inductive hybrid cell forms between the blocking-up CD4-express cell; (3) external effectively in and the CD4 positive cell is infected by the former generation strain isolated from HIV1 type and all clade of HIV2 type and (4) prevention is infected by former generation strain isolated of HIV.

This peptide combinations for prevention and treatment HIV-1 all clade former generation strain isolated and the HIV that causes of former generation strain isolated of HIV-2 infect, and the immunne response of cd4 cell-mediation of not expecting for treatment for example suppresses to repel and independently Immunological diseases for example rheumatoid arthritis, systemic lupus erythematous and psoriasis are useful.

The accompanying drawing summary

Fig. 1 has described the aminoacid sequence (SEQ ID NO:1) of people CD4, and it is the part of host cell antigens c D4/ coreceptor mixture, and it is inferred by nucleotide sequence.

Amino acid is with following standard single-letter coded representation:

Ala:A??Cys:C?His:H??Met:M?Thr:T?Arg:R??Gln:Q

Ile:I??Phe:F?Trp:W??Asn:N?Glu:E?Leu:L??Pro:P

Tyr:Y Asp:D Gly:G Lys:K Ser:S Val:V numbering system be describe among the Littman etc. (cell, 1988,55:541).Xia Huaxian district (AA27-AA66) is the zone in CD4-CDR2 antigen peptide of the present invention source.

Detailed Description Of The Invention

" 1 type human immunodeficiency virus's (HIV-1) Primary isolate " used herein is by restrictedly being cultured to for the 5th generation and obtaining from the PMBC (PBMC) of donor being upper. Primary isolate can from T clone adaptability (TCLA) the laboratory strain of the several times that people T-lymphocytic series, go down to posterity for example among III b/LAI, SF2 and the MN explanation come out. At first, most of Primary isolates are difficult for growing in T clone, and their performance plasomidums induce (SI) and non--plasomidum to induce (NSI) phenotype. For example, much in the PBMC culture, induce the Primary isolates of Syncytium formation in the especially responsive MT2 T clone of HIV-, to copy, but seldom a part for example copy among CEM or the H9 in objectionable T clone almost. The NSI Primary isolate will only copy in former generation T cell. Secondly, they and TCLA strain difference be they the sensitiveness of the neutralization of external restructuring soluble form (rsCD4) to virus receptor protein CD4 (Daar etc., PNAS USA, 1990,87:6574-6578). The 3rd, laboratory-adaptability strain is to the neutralization sensitivity of the antibody that is specific to peplos, and Primary isolate is resistance (Sawyer etc., Journal of Virology, 1994,68:1342; Mascola etc., the infectious disease magazine, 1996,173:340).

" CD4 " used herein refers to that all are by the CD4 protein of naturally occurring CD4 gene code. Originally CD4 is described as the auxiliary lymphocytic cell surface sign of a kind of T-. Find that afterwards CD4 seldom expresses at monocyte, island cell, microglia cell and B cell constituents. Find that also the CD4 molecule participates in the activation of antigen-specificity T auxiliary cell directly by its function as MHC class Ⅱmolecule acceptor. In 1984, finder CD4 be HIV acceptor (Dalgleish etc., the nature, 1984,312:763). The HIV envelope glycoprotein, the combination of gp120 and CD4 represents the first step of Virus entry target cell. The amino acid sequence of people CD4 is incorporated herein (cell, 1985,42:93 from the document of Maddon etc.; With Littman etc., cell, 1988,55:541), shown in Fig. 1 and SEQ ID NO:1.

" recombinant soluble CD4 " used herein or " rsCD4 " are a kind of recombinant microorganism that is made up of the AA1-AA375 of people CD4 or the polypeptide (Fig. 1, SEQ ID NO:1) of cellular expression.

" surface C D4 compound " used herein or " surface complex that contains CD4 " or " containing the surface receptor of CD4/coreceptor compound " refer to CD4 protein in its natural environment at mammalian cell surface and/or complete self CD4 protein of being combined with each other and occurring with any related membrane protein.

Term used herein " immunogene " relates to a kind of peptide combinations, when it is used the host, can induce the antibody for the sub-site of target effect on the CDR2 domain that is present in CD4 (SEQ ID NOS:2 and 3), produce the high-titer antibody that the Primary isolate that derives from 1 type HIV (HIV-1) and 2 all clade of type HIV (HIV-2) is had extensive neutralization activity. The CDR2-CD4 target site represents with lowerization line among Fig. 1, lists in SEQ ID NOS:2 and 3 simultaneously.

The length of " CD4-CDR2 antigenic peptides " of the present invention is about 25 to 50, between preferred 30 to 46 amino acid, contains two by the separated cysteine residues of insetion sequence of 28 to 40 amino acid residues. Insetion sequence can be any continuous part of the residue 27-66 representative sequence of SEQ ID NO:1, perhaps can be the immunologic function analog of the residue 27-66 of a kind of SEQ ID NO:1.

Peptide bond used herein refers to contain the CD4-CDR2 antigenic peptides of the auxiliary epitope peptide of the covalently bound Th of having, and except the direct peptide of molecule is synthetic, can be undertaken covalently bound by any other method. The CD4-CDR2 antigenic peptides is mercaptan-halogen family acetamide coupling in Th epitope peptide covalent coupling with the example that forms the peptide bond, mercaptan-maleimide coupling, mercaptan-mercaptan interchain disulfide bond formation etc.

" peptide based immunogens " used herein refers to a kind of peptide or peptide bond, and it contains the CD4-CDR2 antigenic peptides of the covalently bound Th of having epitope peptide, the optional immunostimulatory peptides that further contains a kind of routine, the spacer region that a kind of attachment and this paper further describe; It has the ability of the antibody of inducing anti-CD4-CDR2 antigenic peptides.

Term used herein " analog " refers to and the peptide that has substantially the same amino acid sequence up to about 10% amino acid whose conservative replacement. Conservative replacement be one of them amino acid by another 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, a preferably replacement from same amino acid (for example, hydrophobic, polarity, electrically charged etc.), but those replacements of the immunological properties of not obvious change peptide. Analog can manually obtain, and the natural variant that exists that also can be used as peptide sequence disclosed herein occurs.

The immunologic function analog is the substantially the same reaction of induction of immunity system, for example, and T-cell response, B-cell response or induce the analog of the antibody of anti-specific antigen.

The present invention relates to new peptide combinations as immunogenic application. This immunogene is useful for the antibody in the effect site (SEQ ID NOS:2 and 3) on the CDR2 domain of the anti-CD4 that produces high-titer by active immunity in mammal comprises human body. Immunogene of the present invention is for prevention and treatment immunodeficiency virus infection and for the cell-mediated immune response of CD4+ that refers to not expect, for example for example rheumatoid arthritis, systemic loupus erythematosus and psoriasis of graft rejection and autonomous immunological diseases.

The application of this specific C D4-reactive antibody aspect prevention and treatment HIV infection and immunity disease, be a kind of immunization therapy, can be by using the passive acquisition of site-specific " site target " antibody prophylactic treatment on the CDR2-spline structure territory of CD4. As described herein, preferred treatment can be passed through active immunity, namely by realizing with the composition inoculation host of containing one or more peptide based immunogens of the present invention. These immunogenes induce the host to produce self site target CD4-CDR2 reactive antibody, and this antibody has the extensive neutralization activity of the Primary isolate of anti-1 type HIV (HIV-1) and 2 all clade of type HIV (HIV-2). Believe that these active immunities will provide the protection more effective and more long-term than passive immunity.

Target site (SEQ ID NOS:2 and 3) on the CDR2-spline structure territory of people CD4 (SEQ IDNOS:2 and 3) is conformational restriction, namely by adding cysteine residues cyclisation (SEQ ID NOS:4 and 5) at N and C end. This target site can also comprise and contains 1-5 from the SEQ ID NOS:4 of one of SEQ ID NOS:2 and 3 ends plus Amino Acid and 5 immunogenic analog (for example, SEQ ID NOS:10 and 11), as long as remain with the ring structure of single disulfide bond.

By carrier protein is arrived in the target site chemical coupling, for example, on the BEDT-TTF A (PEA) of keyhole limpet hemocyanin, (KLH) and modification, further its modification is become immunogenicity CD4-CDR2 antigenic peptides. Should be based on the defective of the vaccine of " CD4-CDR2 antigenic peptides-carrier protein " the weak immunogenicity in (1) target antigen site, a kind of and nearly all self-built in problem that antigen is relevant; (2) the non-functional antibody of the anti-carrier protein of a big chunk; (3) epi-position of carrier-induce suppresses potentiality.

Therefore by chemical coupling or preferably be defined as by the synthetic series connection of direct peptide additional chemical and mix Th and/or other immunostimulatory peptides, it is preferred giving peptide based immunogens. Preferred immunogene of the present invention makes the generation of uncorrelated antibody reduce to minimum, thereby stimulates the immune response more single-minded to " target sequence ". Desirable antibody has the reactivity with the CD4 surface complex, but does not produce the complicated side effect of not expecting of immunization therapy process that may make prevention and treatment HIV infection and immunity disease. And the site-specific antibodie in the required site of target can mix Th by application and produce more widely in genetic diversity host group. The Primary isolate that these antibody responses produce 1 type and 2 all clade of type HIV has the active high-titer antibody of extensive neutralization.

The invention still further relates to and use described peptide combinations as the immune response of not expecting of immunogene prevention and treatment HIV and treatment cd4 cell-mediation for example graft rejection and autonomous for example rheumatoid arthritis, systemic loupus erythematosus and psoriasis of immunological diseases.

Different with the antibody that is specific to rsCD4, the antibody that is specific to the host cell surface acceptor that comprises CD4/common-receptor complex may be in the following aspects and immune system interaction:

1. they may block the CD4-II class interaction between CD4-expressivity T cell and other active t cell, B cell or the monocyte;

2. they transmit signal may for the T cell, therefore suppress the cell-mediated immunoloregulation function of normal CD4+T-;

3. they may engage the trigger cell apoptosis simultaneously by the φt cell receptor molecule, and induce the cell death of CD4-expressivity;

4. they block the interaction between CD4 and the HIV, and it suppresses the immunopathology of HIV-mediation.

The anti-antibody that contains the surface complex of CD4 is the good candidate thing of prevention and treatment HIV infection and the diseases related AIDS of comprising of HIV-.On a more general level, the antibody of anti-surface C D4 mixture may or be cured the cell-mediated immunne response of not expecting of CD4-expressivity T for prevention, and for example for example rheumatoid arthritis, systemic lupus erythematous or psoriasis are effective for transplant rejection and autonomous Immunological diseases.

The characteristic of the antibody that is produced by peptide combinations of the present invention is summarized as follows based on the result that embodiment 1-5 obtains:

In elisa assay in conjunction with rsCD4;

In immunofluorescence analysis in conjunction with CD4-expressivity cell; With

3. in external microplate is analyzed in the neutralization and resistance-HIV strain isolated of former generation.

Antibody with these features is to be particularly useful in the human diseases that causes of the infector of CD4-positive cell in prevention and treatment major objective.Therefore, the invention provides peptide combinations as immunogen, being used to prevent and treating major objective is diseases related all stages that comprise AIDS of human diseases, particularly HIV-that the infector of CD4-positive cell causes.The present invention also provides the application method of these antibody compositions.

Peptide combinations of the present invention comprises peptide based immunogens, and its insertion is positioned at one of two peptide sequences in respective objects effector site on the CDR2-spline structure territory of CD4 (SEQ ID NOS:2 and 3), or its immunologic function analogue.This is immunogenic be characterised in that they induce CD4/ on the CDR2 structural domain of anti-CD4 common-neutralizing antibody in the sub-site of receptor effect.This immunogen relies on through (1) accurate epitope mapping; (2) because their self conformation, cyclisation is to limit their conformation; (3) their reactive widely Th reply the locus specificity of its optimization of acquisition, induce protection antibody to reply.

Specifically, target site is from the CDR2-spline structure territory of self people CD4 sequence.The aminoacid sequence of people CD4 is from (cell, 1985 such as Maddon; 42:93; With Littman etc., cell, 1988; Document 55:541-) is introduced into this paper, represents in Fig. 1 and SEQID NO:1.The CD4-CDR2 target site and is listed in SEQ ID NOS:2 and 3 shown in the underscore among Fig. 1 part.Peptide combinations of the present invention preferably produces as the synthetic peptide that contains target site (SEQ ID NOS:2 and 3), wherein target compound by or near himself sequence of N terminal and C-terminal inserts cysteine residues and is modified so that the formation (for example SEQ ID NOS:4 and 5) of promotion cyclisation peptide.

Peptide combinations of the present invention also comprises 1-5 the immunogenic analog (for example, SEQ ID NOS:10 and 11) that adds amino acid whose SEQ ID NOS:4 and 5 that may contain from one of SEQ ID NOS:2 and 3 ends, as long as remain with the ring structure of single disulfide linkage.Target site comprises that also containing a length is about 25-50 amino acid, has the immunologic function analogue from the cyclisation peptide of the continuous amino acid sequence of SEQ ID NOS:2 and 3.Ring texture is a fundamental of the present invention because contain the peptide of the linear counterpart of target site can not induce have in and the active antibody of HIV strain isolated of former generation.

In addition, by carrier protein and synthetic immunostimulation material for example being derived from mixing the Th epi-position, manually mixing the Th epi-position and the routine immunization stimulator polypeptide is covalently bound of pathogenic virus and bacterium, give the target site immunogenicity of peptide of the present invention.The specific examples of carrier protein and immunostimulation material is existing to be provided, for example BEDT-TTF A (PEA) carrier protein of keyhole limpet hemocyanin (KLH) carrier protein, modification, the Th (SEQ ID NO:8) that derives from hepatitis B virus surface antigen, artificial T h (for example, SEQ ID NO:6) and the routine immunization that derives from Yersinia stimulate invasion peptide (Inv) (SEQID NO:7).

Synthetic fully peptide of the present invention can be expressed from the next: (A) _n-(Th) _m-(B) _o-(CD4-CDR2 antigen peptide)-X

Or (A) _n(CD4-CDR2 antigen peptide)-(B) _o-(Th) _m-X

Or (CD4-CDR2 antigen peptide)-(B) _o-(Th) _m-(A) _n-X

Or (Th) _m-(B) _o-(CD4-CDR2 antigen peptide)-(A) _n-X

Wherein:

Each A is amino acid, α-NH independently ₂, or routine immunization stimulator polypeptide;

Each B be independently selected from amino acid ,-NHCH (X) CH ₂SCH ₂CO-,-NHCH (X) CH ₂SCH ₂CO (Lys-of ε-N) ,-NHCH (X) CH ₂The S-succinimido (Lys-of ε-N) and-NHCH (X) CH ₂S-(succinimide)-;

X is amino acid whose α-COOH or α-CONH ₂

Th is a kind of helper T cell epitope or a kind of immunostimulant analogue or its fragment;

" CD4-CDR2 antigen peptide " is preferably SEQ ID NO:4 or SEQ ID NO:5 as the definition of front, or its cross reactivity immunologic function analogue;

N is 1 to about 10;

M is 1 to about 4; With

O is 0 to about 10.

Peptide combinations of the present invention contains length and is about 30 to 115 amino-acid residues, preferably is about 40 to 90 amino-acid residues, is more preferably the peptide based immunogens of 50 to 80 amino-acid residues.

If A is a seed amino acid, it can be the amino acid that any naturally occurring or non-natural exists.The amino acid that non-natural exists includes but not limited to D-amino acid, Beta-alanine, ornithine, nor-leucine, norvaline, oxyproline, desiodothyroxine, γ-An Jidingsuan, homoserine, citrulline etc.And if m is an amino acid greater than 1, two or three A groups, each amino acid can be identical or different independently so.

If A is a kind of structural domain of invasin, then it can be from the proteic immunostimulation epi-position of Yersinia kind invasin.This immunostimulatory properties derives from the ability of the beta 1 integrin interaction of molecules that exists on this invasin structural domain and the T cell.The particular sequence of discovery and the interactional invasin structural domain of beta 1 integrin is by description (European Journal of Immunology, 1993 such as Brett; 23:1608).

Described a kind of preferred implementation that is used to connect the invasin structural domain (Inv) that mixes the Th epi-position in US 5759551, it inserts this paper through reference.The Inv structural domain preferably has following sequence: Thr-Ala-Lys--Ser-Lys-Lys-Phe-Pro-Ser-Tyr-Thr-Ala-Thr-Tyr-Gln-Phe

(SEQ ID NO:7) or for deriving from its immunostimulation analogue of another kind of Yersinia kind invasin albumen respective regions.This analogue can also contain replacement, deletion or the variation of strain system, as long as analogue keeps immunostimulatory properties.This invasin structural domain is preferably by a kind of transcribed spacer combination, as long as amino acid " A " is added on the Th peptide.

In a preferred implementation, n is 3, (A) 3 is followed successively by a kind of invasin structural domain (Inv), glycine and glycine.

(B) _oOptional is a kind of transcribed spacer, can comprise the amino acid that natural existence or above-mentioned non-natural exist.Each B is identical or different independently.Carrier protein passes through transcribed spacer (for example, Lys-Lys-Lys) through chemical coupling and peptide covalent attachment.(B) _oAmino acid can also be a kind of transcribed spacer that mixes between Th epi-position and the CD4-CDR2 antigen peptide (SEQ ID NOS:4 and 5) that is positioned at, and for example Gly-Gly or ε NLys are so that induce effective antibody response.Except making Th epi-position and B cell epitope (for example SEQ ID NOS:4 and 5) and its immunogenic analog physical separation, a kind of transcribed spacer for example Gly-Gly can destroy the Th epi-position combines generation with the CD4-CDR2 antigen peptide all artificial secondary structures, has therefore weakened the interference between Th and/or the B cell response.

(B) _oAmino acid can also constitute a kind of transcribed spacer as the snappiness hinge that strengthens Th and IgE structural domain compartmentation.In the heavy chain immunoglobulin hinge region, found the example of the sequence of coding snappiness hinge.The common proline rich of snappiness hinge sequence.A kind of useful especially snappiness hinge is sequence Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ IDNO:9), and wherein Xaa is an arbitrary amino acid, is preferably aspartic acid.By (B) _oThe conformation that amino acid provides makes the interaction between peptide based immunogens of the present invention and suitable Th cell and the B cell more effective at interval, has strengthened the immunne response to Th epi-position and antibody-inducibility epi-position and their cross-interaction and immunologic function analogue thus.

Th is a kind of aminoacid sequence (natural or alpha-non-natural amino acid) of the Th of containing epi-position.The Th epi-position can be made of continuous or discontinuous epi-position, therefore is not that each amino acid of Th must be the part of epi-position.The Th epi-position comprises the analogue and the fragment of Th epi-position, can strengthen or stimulate to the CD4-CDR2 antigen peptide (for example, SEQ ID NOS:4 and 5, with and the immunologic function analogue) immunne response.Show as immune structure territory and miscellaneous Th epi-position and in animal and human group, have height and reactive widely (Partidos etc., 1991 with divergent evolution MHC type widely; US 5759551).The Th structural domain of peptide of the present invention has 10 to 50 amino acid approximately, preferably has 10 to 30 amino acid approximately.When multiple Th epi-position (being m 〉=2) occurring, then each Th epi-position is identical or different independently.The Th fragment is the sequential portion of Th epi-position, and it is enough to strengthen or stimulates CD4-CDR2 antigen peptide (for example, SEQID NOS:4 and 5), and/or to the immunne response of its immunologic function analogue.

Those epi-positions that derive from foreign pathogens and provide as embodiment are provided epi-position of the present invention, but be not limited thereto hepatitis B virus surface and cAg helper T cell epitope (HBsTH and HBc Th), Toxins, pertussis helper T cell epitope (PT Th), tetanus toxin helper T cell epitope (TT Th), Measles virus F albumen helper T cell epitope (MV _FTh), chlamydia trachomatis major outer membrane albumen helper T cell epitope (CT Th), diphtheria toxin helper T cell epitope (DT Th), plasmodium falciparum ring sporophyte helper T cell epitope (PF Th), schistosoma mansoni trisaccharide phosphoric acid salt isomerase helper T cell epitope (SM Th), and intestinal bacteria TraT helper T cell epitope (TraT Th).SEQ ID NOS:2-9 and the 42-52 of pathogenic agent deutero-Th in US 5759551 lists; The auxiliary site P11 of chlamydozoan is at Stagg etc., immunology, and 1993, list among the 79:1-9; HBc peptide 50-69 is at Ferrari etc., Journal of Clinical Investigation, and 1991, to list among the 88:214-222, they are through with reference to inserting this paper, and SEQIDNOS:8,13,38-58 (table 8) list in this article together.

The Th epi-position further comprises artificial idealized Th, for example, and SEQ ID NOS:6,12,36,59 (table 9), and immunologic function analogue.The functional analogue of Th comprises the fragment of immunostimulant analogue, cross reactivity analogue and these Th epi-positions.Functional Th analogue further is included in and conservatively replaces, adds, deletes in the Th epi-position and insert 1 to about 10 amino-acid residues, and it does not change the Th stimulatory function of Th epi-position basically.

Peptide binding substances of the present invention also comprises and a kind of carrier protein (for example keyhole limpet hemocyanin) link coupled CD4-CDR2 antigen peptide (for example SEQ ID NOS:4 or 5).

Preferred peptide of the present invention is to contain CD4-CDR2 antigen peptide (for example SEQ ID NOS:4 or 5, or its immunologic function analogue) and Th epi-position and randomly contain a kind of routine immunization to stimulate the site, for example, and Inv (SEQ ID NO:7).One more preferred embodiment in, the TH epi-position is a kind of HBs Th, HBc Th, MV that derives from foreign pathogens _FTh, PT Th, TT Th, CT Th or HIV Th or a kind of Utopian artificial T h or their immunologic function analogue.Randomly, A is a kind of routine immunization stimulator polypeptide, and for example, Inv (SEQ ID NO:7) is preferably through Gly-Gly or the combination of ε NLys transcribed spacer.

The structure of decorating site perhaps derives from the analogue sequence of another species based on the peptide sequence (the amino acid 27-66 of SEQ ID NO:1) in the CDR2-spline structure territory of taking from people CD4.Described CD4-CDR2 target site is accepted following modification:

(1) adding near the N-end side or inserting a cysteine residues,

(2) near the C-end side, preferably or near 66 or similar position add or insert a cysteine residues and

(3) between the halfcystine that keeps, form a disulfide linkage, thereby produce a ring texture.

Described peptide structure can also comprise the additional amino acid of one of 1 to 5 27-66 that takes from CD4 or 39-66 fragment end, as long as keep single disulfide linkage ring texture (for example, SEQ IDNOS:10 and 11).Preferably, in self sequence the unexpected any insertion halfcystine that is used for cyclisation will by, Serine for example, the conservative replacement.

For example, people CD4-CDR2 target site (SEQ IDNOS:2 and 3) by or (for example add halfcystine near N-and C-are terminal, SEQ ID NOS:4 and 5), perhaps (for example replace by adding a halfcystine and near C-terminal, carry out a halfcystine at the N-end, 67 Phe is substituted by Cys, obtains SEQ ID NOS:10 and 11) the mode cyclisation.Modification, cyclisation and eclipsed CD4-CDR2 antigen peptide with following sequence provides by the mode of embodiment:

Cys His Trp Lys Asn Trp Asn Gln Ile Lys Ile Leu Gly Asn Gln Gly Ser Phe Leu Thr Lys Gly Pro Ser Lys Leu Asn Asp Arg Ala Asp Ser Arg Arg Ser Leu Trp Asp Gln Gly Asn Cys ( SEQ ID NO.:4 ) Cys Asn Gln Gly Ser Phe Leu Thr Lys Gly Pro Ser Lys Leu Asn Asp Arg Ala Asp Ser Arg Arg Ser Leu Trp Asp Gln Gly Asn Cys ( SEQ ID NO.:5 ) Cys His Trp Lys Asn Trp Asn Gln Ile Lys Ile Leu Gly Asn Gln Gly Ser Phe Leu Thr Lys Gly Pro Ser Lys Leu Asn Asp Arg Ala Asp Ser Arg Arg Ser Leu Trp Asp Gln Gly Asn Cys Pro Leu Ile Ile ( SEQ ID NO.:10 ) Cys Asn Gln Gly Ser Phe Leu Thr Lys Gly Pro Ser Lys Leu Asn Asp Arg Ala Asp Ser Arg Arg Ser Leu Trp Asp Gln Gly Asn Cys Pro Leu Ile Ile ( SEQ ID NO:11 ) ,。

The peptide based immunogens inductive antibody by containing CD4-CDR2 antigen peptide of the present invention and the surface receptor that the contains CD4/coreceptor mixture cross reaction of host cell, and in and former generation strain isolated of HIV.The respective objects antigenic site of the cell surface CD4 of other species equally also can derive from the homologous fragment of relevant species.

The cross reactivity of CD4-CDR2 antigen peptide and immunologic function analogue (SEQ IDNOS:4,5,10 and 11) may further include conservative replace, add, deletion or insert one to four amino-acid residue, as long as this peptide analogs can be induced with CD4-CDR2 (SEQ IDNOS:2,3,4 and 5) cross reactivity and in having and the active immunne response of HIV strain isolated of former generation.Conservative replacement, interpolation and insertion are known for those skilled in the art, and can realize easily with non--natural amino acid natural or that this paper limits.

Preferred peptide based immunogens of the present invention is to contain the peptide that following composition divides: (1) is referred to as the ring-type of CD4-CDR2 antigen peptide, the CD4-CDR2 site of modification (for example, SEQID NOS:4,5,10 and 11) or its immunogenic analog and (2) epitope peptide herein.Preferred peptide based immunogens is those cascaded structures that contain cyclisation CD4-CDR2 antigen peptide (SEQ ID NOS:4,5,10 and 11) or its cross reactivity and immunologic function analogue; A kind of transcribed spacer (for example, Gly-Gly or ε NLys); A kind of Th epi-position (for example, SEQ ID NOS:8,38-50,55) that is selected from HBs Th, HBc Th, MVF Th, PT Th, TT Th, SMTh, HIVTh, a kind of artificial T h (for example, SEQ ID NOS:6,12,36,59) or a kind of its analogue; Randomly, a kind of Inv structural domain (SEQ ID NO:7) or its analogue.

Peptide based immunogens of the present invention can be by chemical synthesis preparation known to a person of ordinary skill in the art.Referring to, for example, Fields etc., " synthetic peptide: users' guidebook " chapter 3, Grant, W.H. compiles, Freeman and Co., New York, NY, 1992, p.77.Therefore, can use solid-phase synthesis--mate the Merrifield technology automatically, with t-Boc or Fmoc compound protection α-NH2, and the synthetic peptide of the amino acid of the commercial side chain protected that provides of application.The example that is used for peptide synthetic appropriate device is 430A or 431 type applying biological system peptide synthesizers (the Applied Biosystems peptide Synthesizer Models 430A or 431).

After finishing the assembling of required peptide based immunogens,, excising peptide, and make the functional group on the amino acid side chain take off sealing from resin according to the standard program process resin.By HPLC purifying free peptide, by for example amino acid analysis, mass spectrum and/or the evaluation biochemical character that passes through to check order.The purifying of peptide and characterized method are known for those skilled in the art.

Other chemical process that produces the peptide works in CD4 of containing of the present invention and Th site comprises that a kind of by forming " thioether " key is connected halogen family acetylize and cysteinyl peptide.For example, a halfcystine can be added to the C-terminal that contains the Th peptide, the sulfenyl of halfcystine can be used for electrophilic group with the N-end that is attached to the CD4-CDR2 antigen peptide form a covalent linkage (for example, a halfcystine N-(chloracetyl) or one come from the α of maleimide-or ε-NH2 yl).In this pattern, contain Th-(B) _o-(CD4-CDR2 antigen TAI) or its are reverse, (CD4-CDR2 antigen peptide)-(B) o-Th, being with or without routine immunization stimulates the peptide binding substances composition in site to obtain, wherein one of connector " B " be Gly-Gly, (Lys of ε-N) ,-NHCH (X) CH ₂SCH ₂CO-,-NHCH (X) CH ₂SCH ₂CO (Lys-of ε-N) ,-NHCH (X) CH ₂The S-succinimide (Lys-of ε-N), perhaps-NHCH (X) CH ₂S-(succinimide)-.

Immunogen of the present invention can also be a polymeric.Can realize by immunogen and linking agent being utilized the ordinary method reaction, for example by glutaraldehyde and lysine residue-NH ₂Between reaction.

By another kind of method, promptly make synthetic peptide based immunogens polymerization or copolymerization by using an additional cysteine residues that adds immunogen N-end to.The sulfydryl of N-terminal cysteine can be used for amino acid or a kind of ramose poly-lysyl core element (for example, the K that is incorporated into the modification of halogen family ethanoyl ₂K, K ₄K ₂K or K ₈K ₄K ₂The α in the dimaleoyl imino source of the terminal lysine residue of N-K)-or ε-NH ₂Base forms a kind of " thioether " key.Can also make immunogen polymerization of the present invention become a kind of branched structure (Wang etc., science, 1991 by directly synthetic required peptide structure on a kind of ramose poly lysyl core resin; 254:285-288).

Perhaps, long synthetic peptide based immunogens can be synthetic by known recombinant DNA technology.Some manuals of standards about dna technique provide the detailed method for preparing peptide of the present invention.In order to make up the gene of code book invention peptide, translate into a kind of nucleotide sequence with aminoacid sequence is reverse, the preferred utilization is applied to the organic optimizing codon that gene will be expressed therein.Then, prepare synthetic gene, pass through the overlapping oligonucleotide of composite coding peptide and any essential controlling element usually.Synthetic gene is inserted suitable cloning vector, obtain recombinant chou and identification mark.Expression of peptides under the felicity condition that the expression system of selecting and host are fit to then.

Above-mentioned nucleic acid self can be as the composition that is referred to as " dna vaccination ".In described embodiment of the present invention, by the nucleic acid of the described peptide of will encoding, the codon and the promotor of advantageous applications optimization expression in the human cell import patient self cell, induce immunogenic peptide of the present invention to express in those cells.The method of preparation and application dna vaccination is disclosed in US patent 5580859,5589466 and 5703055; Simultaneously referring to WO97/02840 and W.McKonnell and F.Askari, New England Journal of Medicine, 1996,334:2-45, they are all through by with reference to inserting this paper.The method of thinking preparation and using peptide of the present invention and peptide binding substances within the scope of the invention.

The effect of peptide combinations of the present invention can be by the injection animal for example, cavy, and by checking in the immune serum and the ability of HIV strain isolated of former generation, monitoring is to the hormone immunity RACK response acknowledge of CD4-CDR2 antigen peptide, as the detailed description among the embodiment then.

Another aspect of the present invention provides a kind of vaccine composition that contains a kind of one or more peptide based immunogens of the present invention of immune significant quantity in the acceptable transfer system of a kind of medicine.This immunogenic composition is used to prevent and treat immunodeficiency virus infection and is used for the treatment of for example for example rheumatoid arthritis, systemic lupus erythematous and psoriasis of transplant rejection and autonomous Immunological diseases of the CD4-expressivity T cell-mediated immune responses do not expected

Therefore, can use the composition that routine provides in adjuvant, emulsifying agent, drug acceptable carrier or other vaccine composition peptide combinations of the present invention is mixed with a kind of immunogenic composition.Adjuvant of the present invention or emulsifying agent be can be used for and alum, incomplete Freund's adjuvant, liposyn, saponin, squalene, L121, emulsigen, single phosphatidyl lipid A (MPL), dimethyl-two-octadecyl bromination ammonia (DDA), QS21, ISA206 and ISA 720 comprised, and other known effective adjuvant and emulsifying agent.Those of ordinary skills can easily determine described preparation, described preparation also comprises quick-release and/or sustained release preparation, and is used for the inducible system immunity and/or induces the preparation of local mucous membrane immunity, and it can pass through, for example, immunogen is sealed or is realized with the particulate co-administered.Vaccine of the present invention can be by any conventional route administration, comprises subcutaneous, oral, intramuscular or other non-enteron aisle or enteron aisle administration.Equally, immunogen can be used as single dose or multiple dose administration.Those of ordinary skills can easily determine immunization protocol.

Immunogenic composition of the present invention contains a kind of one or more peptide based immunogens of the present invention and a kind of medicine acceptable carrier of significant quantity.Composition in a kind of suitable dosage unit forms contains the immunogen of the about 0.5 μ g of per kilogram of body weight to about 1mg usually.If with multiple dose administration, can be divided into the appropriate amount of every dosage unit preparations easily.For example, predose, 0.2-2.5mg for example, the immunogen of being represented by peptide combinations of the present invention of preferred 1mg will be used repeated doses (enhancing amount) then by injecting preferred intramuscular injection administration.Dosage will be decided according to patient's age, body weight and general health situation, as vaccine and treatment field are known.

The vaccine that contains the peptide based immunogens mixture of the present invention of two or more Th epi-positions can be in enhancing immunity effect among the crowd widely, and a kind of immunne response of the improvement to CD4-CDR2 antigen peptide (for example, SEQ ID NOS:4 and 5) is provided thus.

Can be to the immunogenic immunne response of synthetic CD4-CDR2 of the present invention by being captured in (vaccine, 1991 such as O ' Hagan; 9:768) describe in the biodegradable particulate of type or send improvement on it.Can with immunogen with or do not seal with adjuvant, described particulate can deliver a kind of immunostimulation adjuvant.Particulate can also with together administration of peptide based immunogens, reply with booster immunization, comprise being specially adapted to for example HIV local mucous membrane immunity of transporting of virus through mucous membrane, and be provided for the time-delay of oral administration and topical or the time-sustained release of periodically replying (O ' Hagan etc., 1991; With Eldridge etc., 1991; 28:287).

In order to understand the present invention better, set forth the following example.These embodiment are explanation for example only, does not constitute any type of restriction to the scope of the invention.

Embodiment

The target antigen site peptide of following examples is synthetic by the solid phase method of describing among the embodiment 1.Every kind of peptide can be expressed from the next: (A) _n-(Th) _m-(B) _o-(CD4-CDR2 antigen peptide) or (A) _n-(CD4-CDR2 antigen peptide)-(B) _o-(Th) _m, but above-described other general formula also is included within the present invention.CD4 target antigen site is a kind of cyclic peptide, by the explanation of SEQ IDNOS:4,5,10 and 11 examples, but comprise that length is about 25-50 amino acid, the immunologic function analogue of cyclic peptide with additional 5 aminoacid sequences of continuous amino acid sequence deriving from SEQ ID NOS:2 or 3 and N-who is combined in ring texture or C-end also should be within the scope of the present invention.

The every kind of peptide that is used for these embodiment has Gly-Gly or Nlys as (B) o transcribed spacer between the CD4-CDR2 target site immunity element of Th and modification, some inserts a kind of optional Inv-Gly-Gly that contains, and (wherein Inv (SEQ ID NO:7) and antigen peptide are (for example, SEQID NOS:32-35) (A) 3 elements coupling), but peptide of the present invention (for example also contains other transcribed spacer, SEQ ID NO:9, NLys) or do not have a transcribed spacer.The Th epi-position, as the example in the table 8, comprise and derive from for example artificial T h (for example SEQ ID NOS:6,12,36,59) shown in proteic other Th epi-position that mixes demonstration in auxiliary site and the table 8 of hepatitis B virus surface and cAg and Measles virus F (SEQ IDNOS:8,13 and 43-58) and the table 9 of foreign pathogens.The peptide of these embodiment comprises that also a kind of optional routine immunization stimulates site (for example SEQ ID NO:7).In addition, the invention is not restricted to Inv as additional immunostimulation element.

Embodiment 1

The design of the evaluation A. peptide in the sub-site of potential effect on the surface C D4/ coreceptor mixture

Site in all four kinds of structural domains of people CD4 and the coreceptor site of representing the external structure territory of four kinds of Chemokine Receptors comprise that CC-CKR1, CC-CKR2b, CC-CKR3, CCKR5 and LESTR are selected the stand-in as peptide.Because " epi-position " of identification is a kind of native configurations (WO97/46697), therefore the linear peptide that derives from above-mentioned acceptor/coreceptor all not with MAb B4 kickback, though when existing some to derive from the peptide of the chemokine coreceptor structural domain shown in the WO97/46697, the reactivity of MAb B4 and rsCD4 is significantly strengthened.

Although MAb B4 and any single CD4-or chemokine coreceptor source peptide lack strong reaction, but detect MAb B4 to deriving from many kinds of zones of CD4 (AA1-A20, AA81-92, AA60-AA109, AA118-AA165, AA235-251, AA297-AA351, or AA361-AA375) weak reactivity.The research that this has pointed out a kind of different aided design to synthesize peptide, it will induce the adjacent site, conformation site to MAb B4 identification to have reactive height affinity antibody, be used for suppressing the HIV infection of target cell.

Therefore the potential site sequence in the external structure territory of all the four kinds of structural domains of this CD4 of spreading all over and multiple chemokine coreceptor will be designed, with be synthesized as target peptide, and be connected with target site and make up peptide (as shown in Table 1 and Table 2), synthetic immunogen by the Th that mixes that will derive from HBsAg (SEQ ID NO:8) and Inv (SEQ ID NO:7).Specific CD4 in these structural domains is selected for cyclisation, and it exposes the 3-dimension model prediction (http:www.pdb.bnl.gov/pdb.bin/pdbids) of ring about the surface of people CD4 based on Brookhaven.Specific ring-type restriction is arranged in these peptides, so that the cross reaction between target antigen site and the natural CD4 molecule is maximized.

Therefore, the several composite structures in the table 1 and 2 are synthetic with the halfcystine that not have in the native sequences to find, introduce, the disulfide linkage ring in the ring structure stand-in of being predicted with generation Brookhaven model.In some cases, replace naturally occurring halfcystine, form the disadvantageous configuration of this model so that prevent with Serine.For chemokine coreceptor source peptide, crosslinked in their natural structure stand-in between external structure territory 2 and 3 peptides is shown in table 2 rightmost, by the cysteine residues formation that exists naturally in the structural domain separately.

The site of table 1 description bar acceptance of the bid * has been designed has specific cyclisation.Other peptide site is linear.The independent CD4 or the CCKR target antigen site of peptide representative of table 1 and 2 configuration hurdle acceptance of the bid " a ".They are used as the substrate antigen based on the ELISA of peptide.The peptide of mark " b " is synthesized the target antigen site that is connected in series with HBs Th site (SEQ ID NO:8) as shown in the figure.The peptide of mark " c " be shown in table 1 and 2 with the varient of immunostimulatory peptides (the SEQ ID NO:7) synthetic of connecting " b " construct of Inv structural domain.The peptide that is designed to " d " is and the 2nd Th peptide that is incorporated into the N-end by a Gly-Gly johning knot varient of CT P11 TH (SEQ ID NO:13) series connection synthetic " b " construct.The peptide of mark " e " is synthesized some " b " that become the target antigen site with the TH site that is positioned at C-terminal and construct N-end.A kind of directly synthetic branch four poly-peptides on the poly-lysine core resin of the peptide representative of mark " g ".The peptide representative of mark " x " contains a kind of peptide of duplex structure, and described duplex structure is formed by connecting by the cysteine residues that the exists naturally formation interchain disulfide bond that is present on the chain separately.

Use in the experiment shown in the table 1 and 2, but artificial T H site ' 1,4,9 PALINDROMIC have been used in other Th site that does not here show " (SEQ ID NO:6) and " SynTh (1,2,4) " (SEQ ID NO:12).Also prepared the peptide that is positioned with the Inv site on the C-terminal, and on the N-terminal (the CD4-CDR2 antigen of CD4-CDR2 antigen peptide-GG-Th-GG-Inv), but do not show.

Also be used to separate the Gly-Gly transcribed spacer in target antigen site and Th site, and the transcribed spacer of separating Th and Inv or the 2nd Th immunostimulation site, synthesized " b ", " c ", " d ", " e ", " x " and " other " Th immunogenic peptide that are used for research table 1 and 2.The peptide based immunogens that screening obtains obtains having the candidate target antigen site of inducing the antibody ability with following properties in the host of immunity:

1. combining target antigen site in elisa assay;

2. in elisa assay, under the situation of the antigen peptide in CD4 source in conjunction with rsCD4,

3. the T cell that in immunofluorescence analysis, contains cell surface receptor/coreceptor mixture of CD4 in conjunction with expression; With

4. in external microplate is analyzed in the neutralization and resistance-HIV strain isolated of former generation.B. the target antigen peptide in the screening of candidate target antigen peptide: 1.CD4-and Chemokine Receptors-source is synthetic

List in the table 1 and 2, its respective configuration goes up and uses the Fmoc chemosynthesis at AppliedBiosystems automatic peptide synthesizer (430,431 and 433A type) by the Merrifield solid phase synthesis technique respectively for the peptide of " a ", " b ", " c ", " d ", " e " or " x ".By specified adequate rate in the design that SEQ ID NO:6 is provided, be used at the replaceable aminoacid mixture of particular variable position link coupled, preparation contains a kind of peptide based immunogens that is used for the structure synthetic antigen library (SSAL) of artificial T cell epitope " (1,4,9 PALINDROMIC) Th " (SEQ ID NO:6).SSAL peptide with library of designing for B cell target antigen site or other SSAL TH site can synthesize in a similar manner.After the assembling of finishing these required peptides, use the trifluoroacetic acid process resin according to standard program, excising peptide, and make the blocking group on the amino acid side chain take off sealing from resin.For cyclic peptide, the peptide that excises was left standstill in the 15%DMSO aqueous solution 48 hours, to promote the formation of disulfide linkage between the halfcystine in the chain.By the peptide of HPLC purifying, by mass spectrum and reversed-phase HPLC identification mark through excision, extraction and cleaning.1.CD4 and the target antigen in the Chemokine Receptors-source site-functional effect of specific immunity serum and the formation of evaluation

Estimate the immunogenicity effect of peptide combinations according to the indication of the experiment immunization scheme of antibody response serological analysis as described below.The standard test design:

Immunogen:

(1) individual peptides immunogen; Or

(2) mixing of peptide based immunogens molar ratio, that each scheme is indicated such as contain

Thing.Dosage:

Contain 100 μ g among each immune 0.5 ml, except as otherwise noted.Approach:

Intramuscular, except as otherwise noted.Adjuvant:

(1) Freund's complete adjuvant (CFA)/Freund (IFA);

(2) 0.4% alum (aluminium hydroxide); Or

(3) other adjuvant as described.Every group of a kind of adjuvant of each immunogen.Dosage:

0,2 and 4 weeks; Or 0,3 and 6 week; Or explanation arranged in addition.The CFA/IFA group exists

0 week was accepted CFA, several weeks accepted IFA what follow.Alum or other are specified assistant

The agent group is all accepted same prescription for all dosage.Get the blood scheme:

0th, 3,6 and 8 weeks, or explanation is arranged in addition.Animal varieties:

Duncan Hartley cavy group number:

3 cavy/group analysis:

Anti--active concrete the ELISA of peptide that is used for every kind of immune serum.The solid phase substrate be corresponding " a " type the target antigen peptide (for example, CD4 target antigen peptide, the peptide in Chemokine Receptors source, etc.).

Blood sampling is processed into serum, storage before tiring with target antigen peptide mensuration by ELISA.2. serum and antibody

In several serological analysis, estimate following serological reaction thing, derive from immune serum or murine or the peopleization monoclonal antibody of cavy.Obtain all guinea pig serum in the target antigen site in anti-rsCD4, CD4-and chemokine coreceptor-source as above-mentioned each time point after immunity.Other serological reaction thing obtains by former research or described outside resources.They are introduced into for purpose relatively once in a while.

For example gp anti--gp120 V3 MN (anti--V3 MN) be from through a kind of serum of collecting corresponding to the hyperimmune cavy of antigenic synthetic peptide from the super variable V3 structural domain of HIV-1 MN gp120 (Wang etc., science, 1991,254:285-288).From with about 10 ¹³The GP that obtains in the hyperimmune cavy of peptidoglycan complex mixture that the SSAL of possibility HIV-1 V3 sequence represents is anti--and gp120 V3 MN library serum compiles antiserum(antisera) (anti--V3 SSAL).The V3 MN and the V3 SSAL immunogen that are used for immune guinea pig are the V3 synthetic peptide based immunogens of multiple-limb, it is used for producing, and to have a neutralization several as (" AIDS Review Study " the 18th chapters such as Walfield, volumes such as Koff, Marcel Dekker:New york, 1993, pp.345-360) described HIV-1 tests the active polyclonal antibody of strain system.

Another kind of anti--gp120 antibody is the recombinant human monoclonal antibody of a kind of IgG1 of being called as b12, its gp120 binding site to CD4 have specificity (anti--gp120 CD4-BS) (Burton etc., science, 1994,266:1024-1027).Produce as the segmental IgG1 b12 of a kind of Fab the library from antibody-phage display of the marrow of long-term asymptomatic HIV-1 seropositivity donor preparation, convert it into a kind of complete human antibodies in the recombinant DNA IgG1 expression vector by being cloned into then.During it is counted as and " golden standard " (Burton etc., the same) of the antibody of diversity HIV strain isolated of former generation.3. the ELISA of anti--peptide

By ELISA (Enzyme Linked Immunoadsorbent Assay), use the 96 holes flat droplet degree dull and stereotyped antibody activity of determining anti--peptide of corresponding " a " type target antigen peptide as the immunosorbent coating.Concentration is that the five equilibrium sample (100 μ L) of the target antigen peptide solution of 5 μ g/ml is incubated 1 hour at 37 ℃.Dull and stereotyped with 3% gelatin/PBS solution sealing, once more in 37 ℃ of insulations 1 hour.Dry then sealing is dull and stereotyped, is used for analyzing.The five equilibrium sample of experiment immunization serum (100 μ L), the extent of dilution with 1: 100 in the diluted sample damping fluid is initial, then ten times of serial dilutions is added in the flat board of peptide coating.Dull and stereotyped 37 ℃ of insulations 1 hour.

Use 0.05%PBS/TWEEN ^_Damping fluid washing plate six times.Add suitably dilution at goat-anti--varietY specificity antibody in conjunction with 100 μ L horseradish peroxidase-labeled in the dilution buffer liquid (phosphate buffered saline buffer that contains 0.5 M NaCl and normal goats serum).Dull and stereotyped 37 ℃ of insulations 1 hour, then as above-mentioned cleaning.The five equilibrium sample (100 μ L) that adds the mphenylenediamine substrate solution then.Time formation color with 5-15 minute adds 50 μ L2N H then ₂SO ₄Stop the enzyme color reaction.Each hole A of record in plate reader ₄₉₂Content.Based on the linear regression analysis of optical density, will be by A ₄₉₂Place 0.5, calculate Log with the optical density inverse ₁₀The ELISA that shows tires.This cutoff is accurate, all is less than 0.15 because analyze the value of dilution normal guinea pig control sample at every turn.4. determine with rsCD4 and with the antibody response ability of CD4-express cell

A. determine the reactivity of anti--CD4 by rsCD4 ELISA

(U.S. biotech company from commercial channels, Cambridge, MA) and obtain the recombinant soluble CD4 (rsCD4) of purifying from NIH (USA) AIDS research and reference reagent project (NIH (USA) AIDS Research and ReferenceReagent Program).100 μ L are put in the every hole of 10mM NaHCO3 damping fluid (pH9.5) with 0.25 μ g/ml rsCD4, and 4 ℃ of incubated overnight, the coating 96-hole droplet degree flat board that obtains is used to carry out rsCD4 ELISA.The gelatin PBS solution of 250 μ L, 3% weight being added in the hole of rsCD4-coating, in 37 ℃ of insulations 1 hour, with the nonspecific proteins matter of sealing binding site, is that the PBS of 0.05%TWEEN 20 cleans 3 times with containing volume percent, dry then.

With contain volume percent be 20% normal goats serum, weight percent be 1% gelatin and volume percent be the PBS of 0.05% TWEEN 20 with 1: 20 volume to volume dilution degree serial dilution immune serum or monoclonal antibody, except as otherwise noted.The dilute sample of 100 μ L is added each hole, make it 37 ℃ of reactions 1 hour.Be the PBS solution wash-out hole six times of 0.05%TWEEN 20 with volume percent then, to remove unconjugated traget antibody.The goat of 100 μ L horseradish peroxidase-labeled that is added in volume percent and is extent of dilution among 1% normal goats serum, the PBS that volume percent is 0.05%TWEEN 20 in each hole and be 1: 1000 is anti--and mouse IgG or goat be anti--cavy IgG, in 37 ℃ of insulations 15 minutes.With volume percent is the PBS wash-out hole 6 times of 0.05%TWEEN 20, to remove unconjugated traget antibody binding substances, containing weight percent with 100 μ L then is that 0.04% mphenylenediamine (OPD) and volume percent are sodium citrate buffer solution (pH5.0) the substrate mixture reaction 15 minutes of 0.12% hydrogen peroxide.Add 100 μ L, 1.0 M H ₂SO ₄Stopped reaction is measured 492nm (A ₄₉₂) optical density.Calculate Log ₁₀The inverse of antibody titer is estimated the end reaction of each laboratory sample, as the linear recurrence of inserting, as the description of anti--peptide ELISA.

A. by the definite reactivity of indirect IF staining to the CD4-express cell

Every hole 0.5 * 10 ⁶The CD4-express cell (for example, the cell of HPB-ALL, MT2 or SUP-T1 clone) in containing the PBS of 1%BSA, cleans twice, with them and specified immune serum or monoclonal antibody, test determined optimum concn afterwards, at room temperature be incubated 45 minutes with each.After the cell and the first dyeing antibody insulation, in same cleaning buffer solution, clean again twice, then with suitable dilution second kind of fluorescein isothiocyanate (FITC)-bonded goat anti--mouse IgG or (FITC)-bonded goat is anti--varietY specificity IgG reagent (Cappel, Malvern PA) at room temperature is incubated 45 minutes together.The cell that is colored cleans in same cleaning buffer solution once more, handles cell by being used for determining that the cell fluorescence photograph and/or the immunofluorescence microscopy of staining cell percentage ratio and stain density carry out fluorometric analysis.

B. indirect fluorescent inhibition analysis

For " the biotinylation monoclonal antibody B4-T cell " of relatively using the indirect IF staining technology in conjunction with inhibition analysis; at first cell and the immune serum that disturbs reagent or suitably dilution are incubated jointly; in same cleaning buffer solution, clean twice then, add biotinylated monoclonal antibody B4 afterwards.By being incubated jointly with the FITC-avidin of suitable dilution subsequently, finish the dyeing that CD4-expresses the T cell, and then clean three times, take a picture by cell fluorescence afterwards or the analysis of high explanation fluorescent microscope.5. determine the virus neutralization by antibody

A. cell

With the human T-cell be MT-2 (ATCC 237) the Dulbecco ' s of as mentioned previously interpolation 15% foetal calf serum improve the Eagle substratum (Hanson etc., the Clinical microorganism magazine, 1990, keep growth in 28:2030-2034).By Ficoll-Hypaque gradient separations (Organon Teknika Corp., Durham, NC) peripheral blood lymphocytes (PBMC) of separation HIV-1 seronegativity donor from the buffy coat unit.(Difco Laboratories, Detroit MI) stimulate the PBMC that obtains with 0.5%PHA-P.After 3-4 days, remove the substratum that contains PHA-P, (Buffalo keeps among RPMI NY) cell for Cellular Products, Inc. adding 15% foetal calf serum, 900 μ g/ml glutamine, microbiotic and 5% interleukin-2.

B. viral

HIV-1 MN is a kind of by National Institutes of Health, Bethesda MD (NIH AIDSResearch and Reference Reagent Program Catalog no.402) provide and keep, as the TCLA strain system of the H9 cell culture of persistent infection, therefrom prepare acellular concentrated stock solution.Cultivate the former generation strain isolated for preparing HIV-1 from patient PBMC altogether by PBMC.Preparation is no more than former generation strain isolated stock culture in 3-5 generation in PBMC, then by centrifugal clarification (Sawyer etc., Journal of Virology, 1994,68:1342-1349).California health service portion, the Carl Hanson of Berkeley CA provides them.

The little plaque neutralization analysis of c.MT-2

Use the serum of serial dilution or the HIV pre-incubation of antibody and fixed amount, infected by HIV-responsive MT-2 cell, and formation subsequently shows the cell monolayer of the little plaque of HIV inductive, determines tiring of HIV-neutralizing antibody.The result quantitatively keeps the score by little plaque.This analysis is only applicable to the SI chorista, be TCLA or former generation strain isolated because the huge synplasm that the representative of little plaque is formed by MT-2 cell and the fusion of HIV-cells infected.This analysis is applicable to the inhibition of estimating viral pair cell and the transmission of cell pair cell; because the inhibition that synplasm forms is from the effect of antibody to HIV particle or HIV-cells infected; that is, this assay determination the inhibition that viral pair cell HIV-inductive merges or cell pair cell HIV-inductive merges.Then the minimizing of the plaque observed plaque of 1 week back by enumerating the propidium iodine staining observe neutralizing effect (referring to, Hanson etc., the clinical microbiology magazine, 1990,28:2030-2034).In this was analyzed, virus and serum or antibody diluted in 50% that compile, the fibrinous human normal plasma of removal, to ignore any non-specific enhancing or retarding effect.C. result

Being used for the candidate CD4-of immunogenicity and preliminary functional study or the target antigen site and the peptide in chemokine coreceptor-source describes at table 1 and 2.

With " b " or " c " type target antigen site such as above-mentioned immune guinea pig, unless explanation is arranged in table 3 and 4 in addition, the immune serum that the 6th after first immunisation or 8 weeks collect is analyzed by the description in anti--peptide ELISA and rsCD4 ELISA such as the program.

As shown in table 3 and 4, the peptide based immunogens in most of CD4-and chemokine coreceptor source has hyperimmunization originality, tires at 2.5 to＞5 Log because they are induced ₁₀Anti--peptide antibody in the scope is except peptide p1590b, p1699b, p1699c and p1700b.(for example contain CD4 acceptor long segment, p1612c, p1678b, p1678c, p1686b, p1697b, p1697b, p1817b, p1889b and p1901b) and the antigen site and highly cross reaction of rsCD4 in the CD4-source of the target site of some cyclisation, shown in tiring as the ELISA of anti--rsCD4 of their corresponding＞3.5 Log10 (referring to table 3, the A2 hurdle).The cross reactivity of every kind of peptidic constructs and rsCD4 is uncertain.And, this rsCD4 cross reactivity do not extend to corresponding host cell surface, peptidic constructs has the CD4 of height CD4 cross reactivity, by only finding that with HPB-ALL or MT2 clone indirect IF staining p1697b and p1901b and CD4 express the T cell and have kickback (table 3, B hurdle).

Comparatively speaking, derive from CD4 target antigen site, have the peptide (for example p1472b, p1472c) of ring texture or derive from the serum of CDR2 structural domain (for example p1403b, p1471c) and CD4 expresses T cell kickback, although the cross reactivity lower (table 3, B hurdle) of they and rsCD4.For chemokine common-acceptor, discovery derives from the serum of peptidic constructs p1990, p1999, p2028, p2047, p2048, p2049, p2087 and p2089, wherein great majority have the sequence from coreceptor 1,3 or 4 structural domains, with " surface receptor/coreceptor mixture " reaction (table 4, B hurdle).

The above results shows with rsCD4 or shows that the cross reactivity of acceptor/coreceptor mixture is a kind of complexity and uncertain phenomenon that it only can be observed the conformational characteristic influence of supposition by experiment.

Also screened the neutralization activity of the anti-HIV-1 B clade strain isolated VL of former generation 135 of the immune serum (initial immunity 6 or 8 weeks of back) that obtains by above-mentioned peptidic constructs by the little plaque neutralization analysis of MT reconnaissance-2.Although the high-titer antibody of existence and rsCD4 or " surface C D4/ coreceptor mixture " cross reaction in some immune serum, this neutralizing antibody does not show conspicuous level (table 3 and 4, C hurdle).

The further evaluation with CD4-expressed inhibition or blocking-up MAb B4 and the CD4 expression T cell bonded ability that the T cell has the immune serum of high brightness immunofluorescence dyeing pattern, so that determine the sub-site of potential effect near the discontinuous site of MAb B4 identification conformational epitope.The possibility of result that obtains from these experiments draws the thinking that designs new peptide based immunogens effectively.Carry out above-mentioned evaluation by the experiment that relates to the inhibition of " MAb B4-T cell " bonded immunofluorescence dyeing.With CD4+ target T cell (for example, MT2 T cell) and the suitably immunogen pre-incubation of dilution (for example 1: 10), then cell and biotinylated MAb B4 and FITC-bonded avidin are incubated jointly according to previously described detailed procedure.

In all immune serums of being estimated, only find to suppress the combination (table 5) of MAb B4 by the serum that produces with the peptide p1471c immunity that derives from the CD4-CDR2 structural domain.Those serum that obtain with the peptide immunity of chemokine coreceptor source do not disturb the combination of " MAb B4-T cell ".Should " MAb B4-T cell " in conjunction with inhibiting shortages may part and antibody that the potential effect site is showed dissatisfactory avidity is relevant, may be single because of the site of target antigen site representative due to the space length of MAb B4 recognition site.

The whole anti-acceptor except one and the hyper-immuneserum of coreceptor peptide all can not suppress combining of MAb B4 and T cell, and do not show the neutralization activity of anti-HIV strain isolated of former generation.Further carry out following trial, based on the position of p1471 in the CD4 sequence, the new peptidic constructs that design has target, to catch the potential effect site on the surface C D4, suppressing research by " combination of MAb B4-T cell " provides clue.

More particularly, the peptide that contains around the CD4-CDR2 structural domain, crosses over the target antigen site of 20-75 amino acids residue according to SEQ ID NO:1 numbering system is designed once more, keep at lay special stress under the situation of this district 3D-structure, by derive from have ring texture, the N-of peptide in the CDR2 district of size between 30-45 amino acid and C-end insertion cysteine residues redesigned other peptidic constructs that covers this district.The aminoacid sequence of representative peptidic constructs that derives from described zone is as shown in table 6.

6 or 8 weeks of initial immunity back are collected immune serum, according to being similar to the method evaluation described in the screening of front.In the target antigen site that 41 quilts are estimated, find p205, p2189, p2190 and p2240 (SEQ ID NOS:4,11,10, with 5), promptly " c " construct (SEQ IDNOS:32-35) can be induced the neutralizing antibody (table 6) of anti-HIV-1 strain isolated of former generation.Strain isolated VL135 (table 6) (Sawyer etc., Journal of Virology, 1994,68:1342-1349) be a kind of representational in and resistance strain isolated of former generation.It is not a kind of can be used in provide apparent but easily mislead male atypical in and susceptibility strain isolated of former generation (D.Burton and J.Moore, natural drug, 1998,4:495-48).Therefore, observed here virus neutralization is not a kind of passivation of the virus that is neutralized easily, and prove the wild strain isolated of HIV is attacked the protective immunity that is produced.The immunogen of also not observed chemical concept in the AIDS research field is induced to be had among the above-mentioned strict HIV and the antibody of function.Although MAb B4 lacks in conjunction with active (as shown in the open text WO97/46697 of the patent application of common pending trial) any C4-CDR2 structural domain site peptide, this paper proves the discontinuously arranged epi-position of CD4-CDR2 target site near the recognition site that constitutes neutralizing monoclonal antibody B4.This recognition site may contain from all four kinds of structural domains of CD4 the peptide site, this may be because the arc character of " surface receptor/coreceptor mixture that comprises CD4 " of MAb B4 identification.This " surface C D4 " is different from the extension 3D-model of well-known rsCD4.It should be noted that only some derives from the target antigen site in CD4-CDR2 district, promptly cross over those (for example, p2057c, p2189c, p2190c and p2240c, SEQ IDNOS:32-35) of broader area and performance position cyclic peptide, induce described neutralizing antibody.

The hyper-immuneserum that embodiment 2 peptide p2057c and p2240c produce shows that many kinds of former generations of anti-widely HIV advance

Change the reactive neutralizing antibody of branch

As shown in table 7, in a parallel model with MAb B4 proof pattern, the hyper-immuneserum that derives from the initial immunity of peptide based immunogens p2057c and p2240c (SEQ ID NOS:32 and 35) back 15 and 12 all blood sample collections all proves to have NAT significant, many kinds of clade of anti-HIV strain isolated of former generation of 90%.The progression of NAT scope for increasing is 1: 20 to 1: 185 for clade D, A, B (DH12), E to the former generation strain isolated (being the immune serum at p2057c, SEQ ID NO:32) of B (VL 135) and clade C; Is 1: 20 to 1: 324 for clade D, B (DH12), A, E to the former generation strain isolated (being the immune serum at p2240c, SEQ ID NO:35) of clade B (VL 135).In order to measure equivalent value, MAb B4 is fixed in the 25.6 μ g/ml-1.54 μ g/ml scopes to the neutralization activity of clade B (former generation strain isolated VL 135) former generation strain isolated of HIV-1 clade D, A, E, C, B (DH12).Comparatively speaking, at the guinea pig serum in N section gp120 V3 zone and can't neutralize in any this class and resistance HIV strain isolated of former generation at the monoclonal antibody of the gp120 CD4 binding site (MAb IgG1b12) of the V3 zone of HIV-1 MN (MAb 50.1) gp120 N section or less variation configuration.As demonstrated in Table 7,1: 20 to 1: 300 extent of dilution of the immune serum of anti-p2057c and anti-p2240c (can provide 90% neutralizing effect to E type strain isolated of former generation at HIVA) is approximately the concentration of MAb B4 in the undiluted serum of about 300 μ g/ml, and (numerical value derives from the equivalent and active serum weaker concn and MAb B4 concentration calculating (that is the active immune serum dilution factor of neutralization of the anti-every kind of corresponding strain isolated of the mean concns of MAb B4 (μ g/ml) x).

Embodiment 3 infects in the serum of effect having the anti-SIVmac251 of protection rhesus monkey,

The mutual relationship of NAT

In the patent application WO97/46697 of common pending trial; by MAb B4 antagonism with the protection effect of the attack experimental evaluation MAb B4 of SIV experimental infection rhesus monkey and in vivo in and the mutual relationship of antibody, this also is at human AIDS experimental animal model commonly used.

In this research, with MAb B4 perfusion rhesus monkey, before processing, attack before, and attack back 1 hour time point and collect serum and be used for estimating.By the rsCD4 immunoassay of the MAb B4 curve of precorrection being determined the serum-concentration of MAb B4 antibody, to find all are accepted all animals of 5mg/kg body weight dosage, the serum-concentration of MAb B4 antibody is all in 30-45 μ g/ml scope.In the monitoring phase in 1 year, 3/4 animal of accepting MAb B4 can be protected and antagonism SIVmac251.Be present in (promptly by the concentration of the MAb B4 in the serum in the attack animal body, 30-45 μ g/ml) estimates concentration (about 300 μ g/ml) far below anti--(CD4-CDR2 antigen peptide) antibody, it is by behind the immunogen composition of a kind of p2057c of containing or p2240c (SEQ IDNOS:32 and 35) the immunity host, has the antibody in the immune serum in the host.

Therefore, with the SEQ of comprising ID NOS:4 of the present invention, 5,10 and 11 or the host of the peptide combinations immunity of the present invention of its analogue can expect to have protective immunity, thereby avoid being infected by the strain isolated of many kinds of clade of HIV.

Embodiment 4

The immunogenicity peptide combinations that comprises a specific admixture artificial T h epi-position

According to the design of (SEQ ID NO:6)-Gly-Gly-(SEQ ID NO:5), synthetic a kind of artificial T h/CD4-CDR2 antigen peptide, and called after SEQ ID NO:60.

The peptide of SEQ ID NO:60 is formulated in ISA 206/DDA.ISA 206/DDA is a kind of oil/aqueous emulsion, (MONTANIDETM ISA 206 is by SEPPIC Inc. wherein dimethyl stearyl brometo de amonio (DDA) to be scattered in MONTANIDETM ISA 206 (30mg/ml), Fairfield, but the oiliness metabolism solution that NJ) provides).Then oily suspensions is emulsifiable in the water-based peptide solution with 1: 1 volume ratio, and its adjusted content of peptides is to be provided at the peptide combinations of required dosage amount in the final 0.5ml prepared product.

In above-mentioned preparation, in the cavy of accepting 100 μ g/ dosage 0,3 and 6 weeks, set up the immunogenicity of SEQID NO:60.By the anti--peptide ELISA that describes among the embodiment 1, utilize SEQ ID NO:5 to determine immunogenicity as the cyclisation target antigen site peptide that is used for the solid phase substrate.Whole six cavys all successfully seroconversion become the ELISA reactivity.

Significantly, find that also SEQ ID NO:60 has hyperimmunization originality and functionally active in a large amount of animals.Preparation contains the immunogenic composition of SEQ ID NO:60 in incomplete Freund's adjuvant (IFA), and 300 μ g/ dosage are administered to pig in the 0th, 3 and 6 weeks by intramuscularly then.Pig that transforms through serum and the serum of taking from for the 8th week by the little plaque neutralization analysis of MT-2 (embodiment 1) test to the neutralizing effect activity of antigen for strain isolated HIV-1 VL135.Pig anteserum sample provides 50% neutralizing effect to the virus with extent of dilution injection in 1: 249; Then provide 90% neutralizing effect to 1: 97 extent of dilution.Therefore, give the macrofauna host a kind of immune response with peptide combinations immunity of the present invention, this reaction comprise antibody to the host cell receptor that comprises CD4 and in and the activity of HIV.

Embodiment 5

Representative peptidic constructs of the present invention

Immunogenic peptide of the present invention shown in the table 10 is complete synthetic construct, and it is synthetic by the solid phase method of describing among the embodiment 1.Every kind of peptide in this table all can be by formula (A) _n-(Th) _m-(B) _o-(CD4-CDR2 antigen)-X represents, but the peptide of disclosed other general formula representative in front should be understood in peptide scope of the present invention.The CD4-CDR2 antigen sequence is SEQ IDNO:4 or 5.Shown immunogenic peptide comprises artificial T h site (as shown in table 9).Every kind of peptide of this embodiment all has the Gly-Gly transcribed spacer between immunogenic elements, but peptide of the present invention also may have other transcribed spacer, for example ε NLY or do not have transcribed spacer.Material and method

, excision synthetic, cyclisation and purifying as of the present invention representative peptidic constructs (SEQID NOS:60,61 and 62) table 10 is listed as described in the embodiment 1.Prepare described peptidic constructs, immune animalcule is for example pig or baboon of cavy or large animal for example, is used to estimate their immunogenicity.With peptide volume be comprising of 0.5ml representative emulsifying agent or adjuvant for example suspend among ISA51, ISA720, DDA or the single phosphatidyl lipid A (MPL).For cavy peptide dosage is 100 μ g, is 300 μ g for pig or baboon, will inoculate in the animal muscle.

As the explanation of table 1, animal is accepted injection in 0,3 and 6 weeks.The rsCD4 ELISA that describes by embodiment 1 estimates initial immunity back 5,8 or other specifies the cross reactivity of week test blood sample to rsCD4, then still according to embodiment 1 described further test in them and the ability of HIV-1 strain isolated of former generation.The result

Representative peptidic constructs all has relevant immunogenicity, and all tested peptides are all induced the intensive site-guiding cross reactivity to corresponding rsCD4, and tiring as Log10 in Anti-Human rsCD4ELISA surpasses shown in 3.5 (table 11).Also observe the immune serum that from cavy, pig and baboon, obtains have in and (for example, effect VL135) of HIV-1 strain isolated of former generation.The functional cross reactivity of this of baboon serum merits attention very much because in the baboon serum and the effect of people HIV strain isolated of former generation be almost a kind of human system.Therefore, this primate model has effectively indicated the effect of peptidic constructs of the present invention as the reagent that infects by active immunity prevention and/or immunotherapy HIV.

Table 1

Derive from the structrual description of the peptide of CD4

Code	Describe	Form+	Code	Describe	Form+
Code	Describe	Form+	Code	Describe	Form+	????-	rsCD4	????a	????p1616	(C)CD4(40-43)(C)?? ^*	????b,c
??p1403	CD4(43-55)	????b,c	????p1617	(C)CD4(52-54)(C) ^*	????b,c	????-	rsCD4	????a	????p1616	(C)CD4(40-43)(C)?? ^*	????b,c
??p1403	CD4(43-55)	????b,c	????p1617	(C)CD4(52-54)(C) ^*	????b,c	??p1404	(C)CD4(38-47)(C) ^*	????b,c	????p1618	(C)CD4(51-55)(C) ^*	????b,c
??p1405	CD4(81-92)	????b,c	????p1619	(C)CD4(48-52)(C) ^*	????b,c	??p1404	(C)CD4(38-47)(C) ^*	????b,c	????p1618	(C)CD4(51-55)(C) ^*	????b,c
??p1405	CD4(81-92)	????b,c	????p1619	(C)CD4(48-52)(C) ^*	????b,c	??p1406	CD4(18-29)	????b,c	????p1620	(C)CD4(85-90)(C) ^*	????b,c
??p1460	CD4(1-109)	????a	????p1621	(C)CD4(85-89)(C) ^*	????b,c	??p1406	CD4(18-29)	????b,c	????p1620	(C)CD4(85-90)(C) ^*	????b,c
??p1460	CD4(1-109)	????a	????p1621	(C)CD4(85-89)(C) ^*	????b,c	??p1468	CD4(47-64)	????b,c	????p1622	(C)CD4(85-90)(C) ^*	????b,c
??p1469	CD4(39-45)	????b,c	????p1678	(C)CD4(291-306)(C) ^*	????b,c	??p1468	CD4(47-64)	????b,c	????p1622	(C)CD4(85-90)(C) ^*	????b,c
??p1469	CD4(39-45)	????b,c	????p1678	(C)CD4(291-306)(C) ^*	????b,c	??p1470	(C)CD4(36-45)(C) ^*	????b,c	????p1679	(C)CD4(297-302)(C) ^*	????b,c
??p1471	CD4(39-67)	????b,c	????p1680	(C)?CD4(303-315)(C) ^*	????b,c	??p1470	(C)CD4(36-45)(C) ^*	????b,c	????p1679	(C)CD4(297-302)(C) ^*	????b,c
??p1471	CD4(39-67)	????b,c	????p1680	(C)?CD4(303-315)(C) ^*	????b,c	??p1472	(C)CD4(115-130(C→S)-137)	????b,c	????p1681	(C)CD4(309-313)(C) ^*	????b,c
??p1518	CD4(60-109)	????b,c	????p1682	(C)CD4(314-328)(C) ^*	????b,c	??p1472	(C)CD4(115-130(C→S)-137)	????b,c	????p1681	(C)CD4(309-313)(C) ^*	????b,c
??p1518	CD4(60-109)	????b,c	????p1682	(C)CD4(314-328)(C) ^*	????b,c	??p1585	(C)CD4(36-47)(C) ^*	????b,c	????p1683	(C)CD4(291-306)(C) ^*	????b,c
??p1586	(C)CD4(42-59)(C) ^*	????b,c	????p1684	(C)CD4(315-346)(C) ^*	????b,c	??p1585	(C)CD4(36-47)(C) ^*	????b,c	????p1683	(C)CD4(291-306)(C) ^*	????b,c
??p1586	(C)CD4(42-59)(C) ^*	????b,c	????p1684	(C)CD4(315-346)(C) ^*	????b,c	??p1587	(C)CD4(44-56)(C) ^*	????b,c	????p1685	(C)CD4(338-343)(C) ^*	????b,c
??p1588	(C)CD4(46-55)(C) ^*	????b,c	????p1686	(C)CD4(341-360)(C) ^*	????b,c	??p1587	(C)CD4(44-56)(C) ^*	????b,c	????p1685	(C)CD4(338-343)(C) ^*	????b,c
??p1588	(C)CD4(46-55)(C) ^*	????b,c	????p1686	(C)CD4(341-360)(C) ^*	????b,c	??p1589	(C)CD4(79-96)(C) ^*	????b,c	????p1687	(C)CD4(349-353)(C) ^*	????b,c
??p1590	(C)CD4(104-115)(C) ^*	????b,c	????p1688	(C)CD4(202-205)(C) ^*	????b,c	??p1589	(C)CD4(79-96)(C) ^*	????b,c	????p1687	(C)CD4(349-353)(C) ^*	????b,c
??p1590	(C)CD4(104-115)(C) ^*	????b,c	????p1688	(C)CD4(202-205)(C) ^*	????b,c	??p1608	CD4(16-25)(C) ^*	????c	????p1689	(C)CD4(213-226)(C) ^*	????b,c
??p1609	(C)CD4(118-128)(C) ^*	????c	????p1690	(C)CD4(217-221)(C) ^*	????b,c	??p1608	CD4(16-25)(C) ^*	????c	????p1689	(C)CD4(213-226)(C) ^*	????b,c
??p1609	(C)CD4(118-128)(C) ^*	????c	????p1690	(C)CD4(217-221)(C) ^*	????b,c	??p1610	(C)CD4(127-130(C→S)-141)(C) ^*	????c	????p1691	(C)CD4(228-239)(C) ^*	????b,c
??p1611	(C)CD4(138-146)(C) ^*	????c	????p1692	(C)CD4(231-237)(C) ^*	????b,c	??p1610	(C)CD4(127-130(C→S)-141)(C) ^*	????c	????p1691	(C)CD4(228-239)(C) ^*	????b,c
??p1611	(C)CD4(138-146)(C) ^*	????c	????p1692	(C)CD4(231-237)(C) ^*	????b,c	??p1612	(C)CD4(133-153)(C) ^*	????c	????p1693	(C)CD4(235-251)(C) ^*	????b,c
??p1613	?CD4(159-169)(C) ^*	????c	????p1694	(C)CD4(243-246)(C) ^*	????b,c	??p1612	(C)CD4(133-153)(C) ^*	????c	????p1693	(C)CD4(235-251)(C) ^*	????b,c
??p1613	?CD4(159-169)(C) ^*	????c	????p1694	(C)CD4(243-246)(C) ^*	????b,c	??p1615	(C)CD4(39-44)(C) ^*	????b,c	????p1695	(C)CD4(247-259)(C) ^*	????b,c
??p1696	(C)CD4(250-255)(C) ^*	????b,c	????p1817	CD4[118-130(C)-159(C)-165] ^*	????b	??p1615	(C)CD4(39-44)(C) ^*	????b,c	????p1695	(C)CD4(247-259)(C) ^*	????b,c
??p1696	(C)CD4(250-255)(C) ^*	????b,c	????p1817	CD4[118-130(C)-159(C)-165] ^*	????b	??p1697	(C)CD4(274-286)(C) ^*	????b,c	????p1818	CD4(81-92))XCD4(6-20)	????bxa
??p1698	(C)CD4(277-282)(C) ^*	????b,c	????p1820	CD4(154-165)XCD4(117-140)	????axb	??p1697	(C)CD4(274-286)(C) ^*	????b,c	????p1818	CD4(81-92))XCD4(6-20)	????bxa

Table 1 (continuous page or leaf)

Code	Describe	Constitute	Code	Describe	Constitute
Code	Describe	Constitute	Code	Describe	Constitute	p1699	CD4(168-182)	b,c	p1821	CD4(154-165)XCD4(123-	a??x??b
p1700	(C)CD4(151-178)(C) ^*	b,c	p1848	CD4[(68-92)-M]	a	p1699	CD4(168-182)	b,c	p1821	CD4(154-165)XCD4(123-	a??x??b
p1700	(C)CD4(151-178)(C) ^*	b,c	p1848	CD4[(68-92)-M]	a	p1701	CD4(361-375)	b,c	p1856	CD4(79-88)XCD4(1-20)	a??x??b
p1702	CD4(14-22)	b	p1857	CD4(79-88)XCD4(1-20)	b??x??a	p1701	CD4(361-375)	b,c	p1856	CD4(79-88)XCD4(1-20)	a??x??b
p1702	CD4(14-22)	b	p1857	CD4(79-88)XCD4(1-20)	b??x??a	p1761	CD4(123-134)	b	p1858	CD4(1-20)	b
p1763	CD4(72-92)	b	P1864	CD4(1-20))XCD4(156-173)	b??x??a	p1761	CD4(123-134)	b	p1858	CD4(1-20)	b
p1763	CD4(72-92)	b	P1864	CD4(1-20))XCD4(156-173)	b??x??a	p1765	(C)CD4(70-84)	b	p1868	CD4(297-351) ^*	b
p1766	CD4(6-26)	b	p1875	CD4(87-98)	b	p1765	(C)CD4(70-84)	b	p1868	CD4(297-351) ^*	b
p1766	CD4(6-26)	b	p1875	CD4(87-98)	b	p1767	CD4(6-20)	b	p1876	CD4(81-98)	b
p1768	CD4(154-165)	b	p1877	CD4(76-98)	b	p1767	CD4(6-20)	b	p1876	CD4(81-98)	b
p1768	CD4(154-165)	b	p1877	CD4(76-98)	b	p1769	CD4(156-173)	b	p1878	(C)CD4(77-98)(C) ^*	b
p1770	CD4(146-170)	b	p1879	(C)CD4(77-103) ^*	b	p1769	CD4(156-173)	b	p1878	(C)CD4(77-98)(C) ^*	b
p1770	CD4(146-170)	b	p1879	(C)CD4(77-103) ^*	b	p1771	CD4(117-140)	b	p1880	CD4(72-103) ^*	b
p1773	CD4(72-92)XCD4(6-26)	a??x??b	p1886	CD4(341-375)	b	p1771	CD4(117-140)	b	p1880	CD4(72-103) ^*	b
p1773	CD4(72-92)XCD4(6-26)	a??x??b	p1886	CD4(341-375)	b	p1775	CD4(72-92)XCD4(6-20)	a??x??b	p1889	CD4(213-251)	b
p1777	CD4(117-140))XCD4	a??x??b	p1901	CD4(1-26)	b	p1775	CD4(72-92)XCD4(6-20)	a??x??b	p1889	CD4(213-251)	b
p1777	CD4(117-140))XCD4	a??x??b	p1901	CD4(1-26)	b	p1778	CD4(117-140))XCD4	a??x??b	p1905	CD4(303-C345-353) ^*	b
p1780	CD4(123-134))XCD4	a??x??b	p2037	(C)CD4(68-C84-88) ^*	b	p1778	CD4(117-140))XCD4	a??x??b	p1905	CD4(303-C345-353) ^*	b
p1780	CD4(123-134))XCD4	a??x??b	p2037	(C)CD4(68-C84-88) ^*	b	p1781	CD4(123-134))XCD4	a??x??b	p2038	(C)CD4(68-C84) ^*	b
p1783	CD4(6-20)XCD4(72-92)	a??x??b	p2078	CD4(1-20)	e	p1781	CD4(123-134))XCD4	a??x??b	p2038	(C)CD4(68-C84) ^*	b
p1783	CD4(6-20)XCD4(72-92)	a??x??b	p2078	CD4(1-20)	e	p1784	CD4(6-26)XCD4(72-92)	a??x??b	p2100	(C)CD4(68-C84-92) ^*	b
p1813	CD4(79-88)	b	p2101	(C)CD4(68-C84-98) ^*	b	p1784	CD4(6-26)XCD4(72-92)	a??x??b	p2100	(C)CD4(68-C84-92) ^*	b

The formation of+peptidic constructs is named as a, b, and c, e, g, and x, wherein: on behalf of target antigen site b, a represent HBsTh-GG-target antigen site c to represent Inv-GG-HBsTh-GG-target antigen site e to represent target antigen site-GG-HBsThg to represent at K ₂The tetramer target antigen site x of branch representative on the KAA core is by the crosslinked peptide chain of interchain disulfide bond ^*By or near the peptide of the halfcystine cyclisation of target antigen site N and C end

Table 2

Derive from the structrual description of the peptide of β Chemokine Receptors

The type of Chemokine Receptors	Represent the peptide antigen in Chemokine Receptors external structure territory
																???????????1			?????????????2			?????????????3			????????????4			???????????????2/3
	Code	Describe	Constitute	Code	Describe	?+	Code	Describe	?+	Code	Describe	?+	Code	Describe	??+	???????????1			?????????????2			?????????????3			????????????4			???????????????2/3
	Code	Describe	Constitute	Code	Describe	?+	Code	Describe	?+	Code	Describe	?+	Code	Describe	??+	CC-CKR1#	p1999	?AA1-AA34	?e	?p2004	AA94- AA107	?e	?p2027	AA168- AA203	?b	?p2028	AA261- AA287	?b	p2006	?AA94-AA107 ?X ?AA172-AA203	?dxa
CC-CKR2#	p2086	?AA1-AA43	?e	?p2087	AA100- AA114	?b	?p2088	AA182- AA207	?b	?p2089	AA269- AA285	?b	p2091	?AA100-AA114 ?X ?AA182-AA207	?bxa	CC-CKR1#	p1999	?AA1-AA34	?e	?p2004	AA94- AA107	?e	?p2027	AA168- AA203	?b	?p2028	AA261- AA287	?b	p2006	?AA94-AA107 ?X ?AA172-AA203	?dxa
CC-CKR2#	p2086	?AA1-AA43	?e	?p2087	AA100- AA114	?b	?p2088	AA182- AA207	?b	?p2089	AA269- AA285	?b	p2091	?AA100-AA114 ?X ?AA182-AA207	?bxa	CC-CKR3#	p2079	?AA1-AA35	?e	?p2080	AA92- AA107	?b	?p2081	AA173- AA204	?b	?p2082	AA265- AA281	?b	p2084	?AA92-AA107 ?X ?AA173-AA204	?bxa
CC-CKR5#	p2045	?AA1-AA29	?e	?p2046	AA88- AA102	?b	?p2047	AA168- AA199	?b	?p2048	AA261- AA277	?b	p2049	?AA88-AA102 ?X ?AA168-AA199	?axa	CC-CKR3#	p2079	?AA1-AA35	?e	?p2080	AA92- AA107	?b	?p2081	AA173- AA204	?b	?p2082	AA265- AA281	?b	p2084	?AA92-AA107 ?X ?AA173-AA204	?bxa
CC-CKR5#	p2045	?AA1-AA29	?e	?p2046	AA88- AA102	?b	?p2047	AA168- AA199	?b	?p2048	AA261- AA277	?b	p2049	?AA88-AA102 ?X ?AA168-AA199	?axa	Fusin (LESTR)#	p1987	?AA1-AA38	?e	?p2041	AA103- AA114	?b	?p1990	AA181- AA203	?e	?p1991	AA262- AA285	?b	p1996	?AA181-203 ?X ?AA106-AA111	?dxa

The formation of+peptidic constructs is made a by row, b, d, e, and x, wherein: on behalf of target antigen site b, a represent HBsTh-GG-target antigen site d to represent CTP11Th-GG-HBsTh-GG-target antigen site e to represent target antigen site-GG-HBsThx representative by two peptide chain # chemokine external structure territory peptides that interchain disulfide bond is crosslinked, infers the numbering system of the aminoacid sequence that from the nucleotide sequence that describes below

LESTR (Loetscher et al, journal of biological chemistry, 1994,269:232)

CC-CKR1, CC-CKR2b, CC-CKR3, CC-CKR5 (M.Samson etc., biological chemistry (Biochemistry) 1996,35,3362)

Table 3

The immunogenicity and the function collection of illustrative plates of the CD4-source peptide of determining by ELISA and neutralization analysis

The peptide code			B	?C	The peptide code	?A1	?A2	?B	?C	The peptide code	?A1	A2	B	C
The peptide code			B	?C	The peptide code	?A1	?A2	?B	?C	The peptide code	?A1	A2	B	C	rsCD4		＞5	+++	＜1∶10	p1588??b	?4.3	?3.2	-	＜1∶10	p1621??c	?3.5	1.1	-	＜1∶10
p1403?b	?4.3	2.6	+++	＜1∶10	p1588??c	?4.3	?3.2	-	＜1∶10	p1622??b	?4.3	1.1	-	＜1∶10	rsCD4		＞5	+++	＜1∶10	p1588??b	?4.3	?3.2	-	＜1∶10	p1621??c	?3.5	1.1	-	＜1∶10
p1403?b	?4.3	2.6	+++	＜1∶10	p1588??c	?4.3	?3.2	-	＜1∶10	p1622??b	?4.3	1.1	-	＜1∶10	p1403?c	?4.3	2.1	Tr	＜1∶10	p1589??b	?4.3	?2.2	-	＜1∶10	p1678??b	?2.8	4.5	+	＜1∶10
p1404?b	?2	＜1	Tr	＜1∶10	p1589??c	?4.3	?2.2	-	＜1∶10	p1678??c	?4.3	4.5	+	＜1∶10	p1403?c	?4.3	2.1	Tr	＜1∶10	p1589??b	?4.3	?2.2	-	＜1∶10	p1678??b	?2.8	4.5	+	＜1∶10
p1404?b	?2	＜1	Tr	＜1∶10	p1589??c	?4.3	?2.2	-	＜1∶10	p1678??c	?4.3	4.5	+	＜1∶10	p1404?c	?4.3	2.6	++	＜1∶10	p1590??b	?4.8	?1.7	-	＜1∶10	p1679??b	?2.5	2.1	-	＜1∶10
p1405?b	?4.3	1.1	-	＜1∶10	p1590??c	?4.8	?1.7	-	＜1∶10	p1679??c	?2.5	1.9	+	＜1∶10	p1404?c	?4.3	2.6	++	＜1∶10	p1590??b	?4.8	?1.7	-	＜1∶10	p1679??b	?2.5	2.1	-	＜1∶10
p1405?b	?4.3	1.1	-	＜1∶10	p1590??c	?4.8	?1.7	-	＜1∶10	p1679??c	?2.5	1.9	+	＜1∶10	p1405?c	?＞5	1.8	-	＜1∶10	p1608??c	?3.3	?0.8	tr	＜1∶10	p1680??b	?4.5	2.9	++	＜1∶10
p1406?b	?4.3	0.9	-	1∶12	p1609??c	?4.3	?1.5	tr	＜1∶10	p1680??c	?4.5	2.5	++	＜1∶10	p1405?c	?＞5	1.8	-	＜1∶10	p1608??c	?3.3	?0.8	tr	＜1∶10	p1680??b	?4.5	2.9	++	＜1∶10
p1406?b	?4.3	0.9	-	1∶12	p1609??c	?4.3	?1.5	tr	＜1∶10	p1680??c	?4.5	2.5	++	＜1∶10	p1406?c	?4.3	0.9	-	＜1∶10	p1610??c	?3.3	?＜1	-	＜1∶10	p1681??b	?4.3	1.8	++	＜1∶10
p1460?a	?4.3	2.4	-	＜1∶10	p1611??c	?2	?1.9	tr	＜1∶10	p1681??c	?3.5	1.6	+	＜1∶10	p1406?c	?4.3	0.9	-	＜1∶10	p1610??c	?3.3	?＜1	-	＜1∶10	p1681??b	?4.3	1.8	++	＜1∶10
p1460?a	?4.3	2.4	-	＜1∶10	p1611??c	?2	?1.9	tr	＜1∶10	p1681??c	?3.5	1.6	+	＜1∶10	p1460?#	3.3	1.2	-	＜1∶10	p1612??c	?＞5	?＞5	-	＜1∶10	p1682??b	?＞5	2.7	+	＜1∶10
p1469?b	4.3	2.6	-	＜1∶10	p1613??c	?4.3	?1.0	tr	＜1∶10	p1682??c	?＞5	2.7	+	＜1∶10	p1460?#	3.3	1.2	-	＜1∶10	p1612??c	?＞5	?＞5	-	＜1∶10	p1682??b	?＞5	2.7	+	＜1∶10
p1469?b	4.3	2.6	-	＜1∶10	p1613??c	?4.3	?1.0	tr	＜1∶10	p1682??c	?＞5	2.7	+	＜1∶10	p1469?c	4.3	3.1	Tr	＜1∶10	p1614??b	?＞5	?1.9	-	＜1∶10	p1683??b	?＞5	1.8	+	＜1∶10
p1470?b	3	0.5	Tr	＜1∶10	p1614??c	?＞5	?2.6	-	＜1∶10	p1683??c	?4.0	1.6	+	＜1∶10	p1469?c	4.3	3.1	Tr	＜1∶10	p1614??b	?＞5	?1.9	-	＜1∶10	p1683??b	?＞5	1.8	+	＜1∶10
p1470?b	3	0.5	Tr	＜1∶10	p1614??c	?＞5	?2.6	-	＜1∶10	p1683??c	?4.0	1.6	+	＜1∶10	p1471?a	3.3	2.5	-	＜1∶10	p1615??b	?2.0	?1.3	-	＜1∶10	p168?4??b	?＞5	2.9	+	＜1∶10
p1471?b	3.3	2.8	Tr	＜1∶10	p1615??c	?4.3	?1.7	-	＜1∶10	p1684??c	?＞5	2.5	++	＜1∶10	p1471?a	3.3	2.5	-	＜1∶10	p1615??b	?2.0	?1.3	-	＜1∶10	p168?4??b	?＞5	2.9	+	＜1∶10
p1471?b	3.3	2.8	Tr	＜1∶10	p1615??c	?4.3	?1.7	-	＜1∶10	p1684??c	?＞5	2.5	++	＜1∶10	?p1471?c	4.3	2.7	++	＜1∶10	p1616??b	?＞5	?1.1	tr	＜1∶10	p1685??b	?＞5	1.8	+	＜1∶10
p1472?b	4.3	1.3	+++	＜1∶10	p1616??c	?3.8	?1.2	-	＜1∶10	p1685??c	?3.0	1.6	+	＜1∶10	?p1471?c	4.3	2.7	++	＜1∶10	p1616??b	?＞5	?1.1	tr	＜1∶10	p1685??b	?＞5	1.8	+	＜1∶10
p1472?b	4.3	1.3	+++	＜1∶10	p1616??c	?3.8	?1.2	-	＜1∶10	p1685??c	?3.0	1.6	+	＜1∶10	p1472?c	4.3	1.8	+++	＜1∶10	p1617??b	?＞5	?1.2	-	＜1∶10	p1686??b	?4.3	4.1	+	＜1∶10
p1518?b	4.3	3.7	-	＜1∶10	p1617??c	?2.0	?1.4	-	＜1∶10	p1686??c	?4.3	3.5	tr	＜1∶10	p1472?c	4.3	1.8	+++	＜1∶10	p1617??b	?＞5	?1.2	-	＜1∶10	p1686??b	?4.3	4.1	+	＜1∶10
p1518?b	4.3	3.7	-	＜1∶10	p1617??c	?2.0	?1.4	-	＜1∶10	p1686??c	?4.3	3.5	tr	＜1∶10	p1518?c	4.3	3.7	-	＜1∶10	p1618??b	?＞5	?1.0	-	＜1∶10	p1687??b	?4.3	2.9	++	＜1∶10
p1585?b	4.3	3.0	-	＜1∶10	p1618??c	?4.3	?1.1	-	＜1∶10	p1687??c	?4.3	2.0	+	＜1∶10	p1518?c	4.3	3.7	-	＜1∶10	p1618??b	?＞5	?1.0	-	＜1∶10	p1687??b	?4.3	2.9	++	＜1∶10
p1585?b	4.3	3.0	-	＜1∶10	p1618??c	?4.3	?1.1	-	＜1∶10	p1687??c	?4.3	2.0	+	＜1∶10	p1585?c	4.3	3.0	-	＜1∶10	p1619??b	?4.3	?1.4	-	＜1∶10	p1688??b	?＞5	2.4	+	＜1∶10
p1586?b	4.3	3.2	-	＜1∶10	p1619??c	?4.3	?1.4	-	＜1∶10	p1688??c	?4.3	2.4	+	＜1∶10	p1585?c	4.3	3.0	-	＜1∶10	p1619??b	?4.3	?1.4	-	＜1∶10	p1688??b	?＞5	2.4	+	＜1∶10
p1586?b	4.3	3.2	-	＜1∶10	p1619??c	?4.3	?1.4	-	＜1∶10	p1688??c	?4.3	2.4	+	＜1∶10	p1586?c	4.3	3.2	-	＜1∶10	p1620??b	?＞5	?1.7	tr	＜1∶10	p1689??b	?4.3	2.9	++	＜1∶10
p1587?b	4.3	3.2	-	＜1∶10	p1620??c	?＞5	?1.4	-	＜1∶10	p1689??c	?3.8	2.5	++	＜1∶10	p1586?c	4.3	3.2	-	＜1∶10	p1620??b	?＞5	?1.7	tr	＜1∶10	p1689??b	?4.3	2.9	++	＜1∶10
p1587?b	4.3	3.2	-	＜1∶10	p1620??c	?＞5	?1.4	-	＜1∶10	p1689??c	?3.8	2.5	++	＜1∶10	p1587?c	4.3	3.2	-	＜1∶10	p1621??b	?＞5	?1.4	-	＜1∶10	p1690??b	?3	2.1	++	＜1∶10

Table 3 (continuous page or leaf)

The peptide code	?A1	?A2	B	?C	The peptide code	A1	?A2	?B	?C
The peptide code	?A1	?A2	B	?C	The peptide code	A1	?A2	?B	?C	?p1690??c	?2.0	?1.9	++	＜1∶10	?p1771??b	＞5	?2.8	+++	＜1∶10
?p1692??b	?4.3	?1.7	+	＜1∶10	?p1773??x	＞5	?1.2	-	＜1∶10	?p1690??c	?2.0	?1.9	++	＜1∶10	?p1771??b	＞5	?2.8	+++	＜1∶10
?p1692??b	?4.3	?1.7	+	＜1∶10	?p1773??x	＞5	?1.2	-	＜1∶10	?p1693??c	?4.3	?2.7	+	＜1∶10	?p1775??x	?4.3	?1.3	-	＜1∶10
?p1694??b	?3.0	?1.8	-	＜1∶10	?p1777??x	?4.5	?1.7	-	＜1∶10	?p1693??c	?4.3	?2.7	+	＜1∶10	?p1775??x	?4.3	?1.3	-	＜1∶10
?p1694??b	?3.0	?1.8	-	＜1∶10	?p1777??x	?4.5	?1.7	-	＜1∶10	?p1694??c	?3	?1.8	Tr	＜1∶10	?p1778??x	＞5	?2.7	-	＜1∶10
?p1695??b	?3.0	?2.0	++	＜1∶10	?p1780??x	＞5	?1.2	++	＜1∶10	?p1694??c	?3	?1.8	Tr	＜1∶10	?p1778??x	＞5	?2.7	-	＜1∶10
?p1695??b	?3.0	?2.0	++	＜1∶10	?p1780??x	＞5	?1.2	++	＜1∶10	?p1695??c	?2.5	?2.1	+	＜1∶10	?p1781??x	＞5	?2.7	-	＜1∶10
?p1696??b	＞5	?1.8	+	＜1∶10	?p1783??x	?4.3	?1.2	-	＜1∶10	?p1695??c	?2.5	?2.1	+	＜1∶10	?p1781??x	＞5	?2.7	-	＜1∶10
?p1696??b	＞5	?1.8	+	＜1∶10	?p1783??x	?4.3	?1.2	-	＜1∶10	?p1696??c	＞5	?1.9	Tr	＜1∶10	?p1784??x	?4.3	?1.4	-	＜1∶10
?p1697??b	＞5	?4.2	++	＜1∶10	?p1813??b	?3.3	?1.5	-	＜1∶10	?p1696??c	＞5	?1.9	Tr	＜1∶10	?p1784??x	?4.3	?1.4	-	＜1∶10
?p1697??b	＞5	?4.2	++	＜1∶10	?p1813??b	?3.3	?1.5	-	＜1∶10	?p1697??c	?3.5	?2.1	+	＜1∶10	?p1817??b	＞5	?4.5	+	＜1∶10
?p1697??c	?3.5	?2.1	++	＜1∶10	?p1818??x	＞5	?1.3	-	＜1∶10	?p1697??c	?3.5	?2.1	+	＜1∶10	?p1817??b	＞5	?4.5	+	＜1∶10
?p1697??c	?3.5	?2.1	++	＜1∶10	?p1818??x	＞5	?1.3	-	＜1∶10	?p1698??b	?3.5	?1.7	-	＜1∶10	?p1820??x	＞5	?2.5	tr	＜1∶10
?p1698??c	?2.0	?1.8	-	＜1∶10	?p1821??x	＞5	?1.3	-	＜1∶10	?p1698??b	?3.5	?1.7	-	＜1∶10	?p1820??x	＞5	?2.5	tr	＜1∶10
?p1698??c	?2.0	?1.8	-	＜1∶10	?p1821??x	＞5	?1.3	-	＜1∶10	?p1699??b	?1.5	?-	-	＜1∶10	?p1848??q	＞5	?3.1	+++	＜1∶10
?p1699??c	?1.5	?-	-	＜1∶10	?p1856??x	?4.3	?1.5	-	＜1∶10	?p1699??b	?1.5	?-	-	＜1∶10	?p1848??q	＞5	?3.1	+++	＜1∶10
?p1699??c	?1.5	?-	-	＜1∶10	?p1856??x	?4.3	?1.5	-	＜1∶10	?p1700??b	?1.5	?-	-	＜1∶10	?p1857??x	＞5	?2.3	-	＜1∶10
?p1700??c	?2.5	?1.7	-	＜1∶10	?p1864??x	?3	?2.5	+++	＜1∶10	?p1700??b	?1.5	?-	-	＜1∶10	?p1857??x	＞5	?2.3	-	＜1∶10
?p1700??c	?2.5	?1.7	-	＜1∶10	?p1864??x	?3	?2.5	+++	＜1∶10	?p1701??b	＞5	?2.2	++	＜1∶10	?p1878??b	?4	?2	-	＜1∶10
?p1701??c	＞5	?2.6	++	＜1∶10	?p1889??b	＞5	?4.4	1.5	＜1∶10	?p1701??b	＞5	?2.2	++	＜1∶10	?p1878??b	?4	?2	-	＜1∶10
?p1701??c	＞5	?2.6	++	＜1∶10	?p1889??b	＞5	?4.4	1.5	＜1∶10	?p1761??b	?4.3	?2.2	++	＜1∶10	?p1901??b	＞5	?4	3	＜1∶10
?p1763??b	?4.3	?2.1	-	＜1∶10	?p2037??b	＞5	?NT	tr	＜1∶10	?p1761??b	?4.3	?2.2	++	＜1∶10	?p1901??b	＞5	?4	3	＜1∶10
?p1763??b	?4.3	?2.1	-	＜1∶10	?p2037??b	＞5	?NT	tr	＜1∶10	?p1765??x	?3.5	?1.3	-	＜1∶10	?p2038??b	?4.3	?NT	-	＜1∶10
?p1766??b	＞5	?1.2	-	＜1∶10	?p2078??b	?4.3	?NT	++	＜1∶10	?p1765??x	?3.5	?1.3	-	＜1∶10	?p2038??b	?4.3	?NT	-	＜1∶10
?p1766??b	＞5	?1.2	-	＜1∶10	?p2078??b	?4.3	?NT	++	＜1∶10	?p1767??b	?3.8	?3.0	-	＜1∶10	?p2100??b	?3.5	?NT	+	＜1∶10
?p1768??b	＞5	?1.4	-	＜1∶10	?p2101??b	?3.0	?NT	tr	＜1∶10	?p1767??b	?3.8	?3.0	-	＜1∶10	?p2100??b	?3.5	?NT	+	＜1∶10
?p1768??b	＞5	?1.4	-	＜1∶10	?p2101??b	?3.0	?NT	tr	＜1∶10	?p1769??b	＞5	?1.3	-	＜1∶10

Legend, table 3:

The #:p1460-BSA binding substances

A1:Log10 ELISA is anti--and the inverse in target antigen site tires

A2:Log10 ELISA is anti--and the inverse of rsCD4 tires

B:IFA (indirect IF staining analysis)

C: in the MT-2 neutralization analysis, the serum dilution when clade B HIV-1 VL 135 50% is suppressed

G: ramose tetramer peptide

Tr: trace NT: do not detect

Table 4

The immunogenicity and the function collection of illustrative plates of the chemokine that obtains by ELISA and neutralization analysis

The type of Chemokine Receptors	Represent the peptide antigen of chemokine receptor domain
	Represent the peptide antigen of chemokine receptor domain																				1				2				3				?4				2/3
	Code	A	B	C	Code	A	B	C	Code	A	B	C	Code	A	B	C	Code	A	B	C	1				2				3				?4				2/3
	Code	A	B	C	Code	A	B	C	Code	A	B	C	Code	A	B	C	Code	A	B	C	CC-CKR1	p1999	＞5	+++	＜1∶10	?p2004	＞5	-	＜1∶10	p2027	2.5	-	＜1∶10	p2028	?4.3	++	＜1∶10	p2006	＞5	-	＜1∶10
CC-CKR2	p2086	4.3	-	＜1∶10	p2087b	4.3	++	＜1∶10	p2088	＞5	-	＜1∶10	p2089	?4.3	+	＜1∶10	p2091	3.5	-	＜1∶10	CC-CKR1	p1999	＞5	+++	＜1∶10	?p2004	＞5	-	＜1∶10	p2027	2.5	-	＜1∶10	p2028	?4.3	++	＜1∶10	p2006	＞5	-	＜1∶10
CC-CKR2	p2086	4.3	-	＜1∶10	p2087b	4.3	++	＜1∶10	p2088	＞5	-	＜1∶10	p2089	?4.3	+	＜1∶10	p2091	3.5	-	＜1∶10	CC-CKR3	p2079	4.3	tr	＜1∶10	p2080??b	3.0	-	＜1∶10	p2081	3.0	-	＜1∶10	p2082	?2.5	tr	＜1∶10	p2084	?4.3	tr	＜1∶10
CC-CKR5	p2045	＞5	tr	＜1∶10	p2046??b	3.0	tr	＜1∶10	p2047	3.5	++	＜1∶10	p2048	?4.8	+++	＜1∶10	p2049	?4.3	++	＜1∶10	CC-CKR3	p2079	4.3	tr	＜1∶10	p2080??b	3.0	-	＜1∶10	p2081	3.0	-	＜1∶10	p2082	?2.5	tr	＜1∶10	p2084	?4.3	tr	＜1∶10
CC-CKR5	p2045	＞5	tr	＜1∶10	p2046??b	3.0	tr	＜1∶10	p2047	3.5	++	＜1∶10	p2048	?4.8	+++	＜1∶10	p2049	?4.3	++	＜1∶10	Fusin (LESTR)	p1987	＞4	tr	＜1∶10	p2041??b	4.3	-	＜1∶10	p1990	2.5	++	＜1∶10	p1991	?3.5		＜1∶10	p1996	＞5	-	＜1∶10

A:Log ₁₀ELISA is anti--target antigen site inverse B:IFA (indirect IF staining analysis) C that tires: and the serum dilution tr that in the MT-2 neutralization analysis, clade B HIV-1 VL 135 50% is suppressed: trace

Table 5

The immune serum that CD4-or chemokine coreceptor source peptide produces to MAB B4 in conjunction with the effect of MT2 T cell inhibiting

+????IFA

# inhibition IFA

* referring to the diagram of table 2 about the peptide code description

Substratum only

Table 6

The ability of the neutralizing antibody of the anti-HIV of inducing peptide strain isolated of former generation

The peptide code	The description in target antigen site	The aminoacid sequence of target antigen peptide (a)	Form	A1	?A2	?B	?C
The peptide code	The description in target antigen site	The aminoacid sequence of target antigen peptide (a)	Form	A1	?A2	?B	?C	?p1403	?CD4(43-55) (SEQ?ID?NO:14)	FLTKGPSKLNDRA	?b	?4.3	?2.6	++	＜1∶10
?c	?4.3	?2.1	tr	＜1∶10							?b	?4.3	?2.6	++	＜1∶10
?c	?4.3	?2.1	tr	＜1∶10	?p1404	(C)CD4(38-47)(C) ^*(SEQ?ID?NO:15)	(C)GNQGSFLTKG(C)				?b	?2	＜1	tr	?NT
?c	?4.3	?2.6	++	＜1∶10							?b	?2	＜1	tr	?NT
?c	?4.3	?2.6	++	＜1∶10				?p1468	?CD4(49-63) (SEQ?ID?NO:16)	GPSKLNDRADSRRSLWDQ	?b	?4.3	?3.1	tr	＜1∶10
?c	?4.3	?2.4	-	＜1∶10							?b	?4.3	?3.1	tr	＜1∶10
?c	?4.3	?2.4	-	＜1∶10	?p1469	?CD4(39-45) (SEQ?ID?NO:17)	NQGSFLT				?b	?4.3	?2.6	-	＜1∶10
?c	?4.3	?3.1	tr	＜1∶10							?b	?4.3	?2.6	-	＜1∶10
?c	?4.3	?3.1	tr	＜1∶10				?p1470	?CD4(37-46)* (SEQ?ID?NO:18)	(C)ILGNQGSFLT(C)	?b	?3	?0.5	tr	＜1∶10
?c	?1.5	?0.8	-	＜1∶10							?b	?3	?0.5	tr	＜1∶10
?c	?1.5	?0.8	-	＜1∶10	?p1471	?CD4(39-67) (SEQ?ID?NO:19)	NQGSFLTKGPSKLNDRADSRRSLWDQGNF				?b	?3.3	?2.8	tr	＜1∶1
?c	?4.3	?2.7	-	＜1∶10							?b	?3.3	?2.8	tr	＜1∶1
?c	?4.3	?2.7	-	＜1∶10				?p1518	?CD4(60-109) (SEQ?ID?NO:20)	SKLNDRADSRRSLWDQGNFPLIIKNLKIEDSDTYICEVEDQKEEVQLLVFGLTANSDTHLL	?b	?4.3	?3.7	-	＜1∶10
?c	?4.3	?3.7	-	?＜1∶10							?b	?4.3	?3.7	-	＜1∶10
?c	?4.3	?3.7	-	?＜1∶10	?p1585	?CD4(36-47) ^*(SEQ?ID?NO:21)	(C)ILGNQGSFLTKG(C)				?b	?4.3	?3.0	-	?＜1∶10
?c	?4.3	?3.0	-	?＜1∶10							?b	?4.3	?3.0	-	?＜1∶10
?c	?4.3	?3.0	-	?＜1∶10				?p1586	?CD4(42-59) ^*????(SEQ?ID?NO:22)	(C)SFLTKGPSKLNDRADSRR(C)	?b	??4.3	??3.2	-	?＜1∶10
?c	??4.3	??3.2	-	?＜1∶10							?b	??4.3	??3.2	-	?＜1∶10
?c	??4.3	??3.2	-	?＜1∶10	??p1587	??CD4(44-56) ^*????(SEQ?ID?NO:23)	(C)LTKGPSKLNDRAD(C)				?b	??4.3	??3.2	-	?＜1∶10
?c	??4.3	??3.2	-	?＜1∶10							?b	??4.3	??3.2	-	?＜1∶10
?c	??4.3	??3.2	-	?＜1∶10				??p1588	??CD4(46-55) ^*????(SEQ?ID?NO:24)	(C)KGPSKLNDRA(C)	?b	??4.3	??3.2	-	?＜1∶10
?c	??4.3	??3.2	-	?＜1∶10							?b	??4.3	??3.2	-	?＜1∶10
?c	??4.3	??3.2	-	?＜1∶10	??p1589	??CD4(79-96) ^*????(SEQ?ID?NO:25)	(C)SDTYICEVEDQKEEVQLL(C)				?b	??4.3	??2.2	-	?＜1∶10
?c	??4.3	??2.2	-	?＜1∶10							?b	??4.3	??2.2	-	?＜1∶10

Table 6 (continuing)

The peptide code	The description in target antigen site	The aminoacid sequence of target antigen peptide (a)	Form	?A1	?A2	??B	?C
The peptide code	The description in target antigen site	The aminoacid sequence of target antigen peptide (a)	Form	?A1	?A2	??B	?C	?p1614	?CD4(38-45)(C) ^*????(SEQ??ID??NO:26)	?(C)GNQGSFLT(C)	??b	?＞5	?1.9	??NT	?＜1∶10
??c	?＞5	?2.6	??NT	?＜1∶10							??b	?＞5	?1.9	??NT	?＜1∶10
??c	?＞5	?2.6	??NT	?＜1∶10	??p1615	??CD4(39-44)(C) ^*????(SEQ??ID??NO:27)	?(C)NQGSFL(C)				??b	?2.0	?1.3	??NT	?＜1∶10
??c	?＞4	?1.7	??NT	?＜1∶10							??b	?2.0	?1.3	??NT	?＜1∶10
??c	?＞4	?1.7	??NT	?＜1∶10				??p1616	??CD4(40-43)(C) ^*????(SEQ?ID?NO:28)	?(C)QGSF(C)	b	＞5	?1.1	??NT	?＜1∶10
?c	?3.5	?1.2	??NT	?＜1∶10							b	＞5	?1.1	??NT	?＜1∶10
?c	?3.5	?1.2	??NT	?＜1∶10	?p1617	?CD4(52-54)(C) ^*????(SEQ?ID?NO:29)	(C)NTR(C)				b	＞5	?1.2	??NT	?＜1∶10
?c	2.9	?1.4	??NT	?＜1∶10							b	＞5	?1.2	??NT	?＜1∶10
?c	2.9	?1.4	??NT	?＜1∶10				?p1618	?CD4(51-55)(C) ^*????(SEQ?ID?NO:30)	(C)LNTRA(C)	b	＞5	?1.0	??NT	?＜1∶10
?c	＞4	?1.1	??NT	?＜1∶10							b	＞5	?1.0	??NT	?＜1∶10
?c	＞4	?1.1	??NT	?＜1∶10	?p1619	?CD4(48-52)(C) ^*????(SEQ?ID?NO:31)	(C)PSKLN(C)				b	＞4	?1.4	??NT	?＜1∶10
?c	＞4	?1.4	??NT	?＜1∶10							b	＞4	?1.4	??NT	?＜1∶10
?c	＞4	?1.4	??NT	?＜1∶10				?p2057	(C)CD4(27-66)(C) ^*????(SEQ?ID?NO:4)	(C)HWKNSNQIKILGNQGSFLTKGPSKLNTRADSRRSLWDQGN(C)	??b	?＞5	??4.4	??NT	?＜1∶10
??c	??4.5	??＞5	??NT	??1∶165							??b	?＞5	??4.4	??NT	?＜1∶10
??c	??4.5	??＞5	??NT	??1∶165	??p2189	??(C)CD4(39-71) ^*??[F ₆₇→C] ????(SEQ?ID?NO:11)	(C)NQGSFLTKGPSKLNDRADSRRSLWDQGN(C)PLII				??c	??＞5	??＞5	??+++	??1∶23
??p2190	??(C)CD4(27-71) ^*??[F ₆₇?→?C] ????(SEQ?ID?NO:10)	(C)HWKNSNQIKILGNQGSFLTKGPSKLNDRADSRRSLWDQGN(C)PL ?II	??c	??＞5	??p2189	??(C)CD4(39-71) ^*??[F ₆₇→C] ????(SEQ?ID?NO:11)	(C)NQGSFLTKGPSKLNDRADSRRSLWDQGN(C)PLII	??＞5	??+++	??1∶15	??c	??＞5	??＞5	??+++	??1∶23
??p2190	??(C)CD4(27-71) ^*??[F ₆₇?→?C] ????(SEQ?ID?NO:10)	(C)HWKNSNQIKILGNQGSFLTKGPSKLNDRADSRRSLWDQGN(C)PL ?II	??c	??＞5	??p2240	??(C)CD4(39-66)(C) ^*????(SEQ?ID?NO:5)	(C)NQGS?ELTKGPSKLNDRADSRRSLWDQGN(C)	??＞5	??+++	??1∶15	??c	??＞4	??＞5	??+++	??1∶283

A1:Log ₁₀ELISA is anti--and target antigen site inverse tires

B:IFA

A2:Log ₁₀ELISA is anti--and the rsCD4 inverse tires

C: the serum dilution that in the MT-2 neutralization analysis, clade B HIV-1 VL 135 50% is suppressed

*: the cyclisation peptide

In table 7 monoclonal antibody and the immune serum and the HIV-1 strain isolated of former generation (the little plaque neutralization analysis of MT-2) of clade A, B, C and E

The antibody type	UGO29# (clade A)	VL 135 (clade B)	ZIM 748# (clade C)	UG266# (clade D)	TH036# (clade E)	DH12# (clade B)
	UGO29# (clade A)	VL 135 (clade B)	ZIM 748# (clade C)	UG266# (clade D)	TH036# (clade E)	DH12# (clade B)	90％	90％	90％	90％	90％	90％
	αp2057c (SEQ?ID?NO:32) (15?wpi)	1∶20	1∶157	?1∶184	1∶20	1∶87	90％	90％	90％	90％	90％	90％	1∶20
?αp2240c (SEQ?ID?NO:35) (12?wpi)	αp2057c (SEQ?ID?NO:32) (15?wpi)	1∶20	1∶157	?1∶184	1∶20	1∶87	1∶76	1∶324	?NT	1∶20	1∶102	1∶36	1∶20
?αp2240c (SEQ?ID?NO:35) (12?wpi)	?MAb?B4	?6.76 ?μg/ml	1.54 μg/ml	?2.82 μg/ml	25.6 μg/ml	3.32 μg/ml	1∶76	1∶324	?NT	1∶20	1∶102	1∶36	?2.1 μg/ml
α N-holds V3 _MN	?MAb?B4	?6.76 ?μg/ml	1.54 μg/ml	?2.82 μg/ml	25.6 μg/ml	3.32 μg/ml	?＞100 ?μg/ml	＞100 μg/ml	＞100 μg/ml	＞100 μg/ml	＞100 μg/ml	＞100 μg/ml	?2.1 μg/ml
α N-holds V3 _MN	?Ig?G1?b12?MAb?α?gp120	?38.5 ?μg/ml	41.7 μg/ml	＞50 μg/ml	＞50 μg/ml	＞50 ?μg/ml	?＞100 ?μg/ml	＞100 μg/ml	＞100 μg/ml	＞100 μg/ml	＞100 μg/ml	＞100 μg/ml	?NT
?MAb?50.1?α?gp120 (N-terminal?V3)	?Ig?G1?b12?MAb?α?gp120	?38.5 ?μg/ml	41.7 μg/ml	＞50 μg/ml	＞50 μg/ml	＞50 ?μg/ml	?＞50 ?μg/ml	＞50 μg/ml	＞50 μg/ml	＞50 μg/ml	＞50 μg/ml	＞50 μg/ml	?NT

#: plan HIV-1 is provided strain isolated of former generation about the whole world of AIDS by WHO.

Table 8

The aminoacid sequence of the Th epi-position in foreign pathogens source

The description of Th	??SEQ?ID?NO:	Aminoacid sequence
The description of Th	??SEQ?ID?NO:	Aminoacid sequence	MV _F288-302?Th MV _F258-277?Th TT _830-844?Th TT _947-966?Th PT _149-176?Th TT _73-99?Th PT _18-41?Th HBs _19-32?Th HBc _120-140?Th HBc _21-40?Th HBc _50-69TT _615-631?Th HIV?gp41?Th ₆(N-) HIV?gp41?Th ₆(C-) CT?A8 _106-130?Th CTP ₁₁Th DT ₁?Th DT ₄?Th PF?Th SM?Th TraT ₁Th TraT ₄Th TraT ₆Th	????38 ????39 ????40 ????41 ????42 ????43 ????44 ?????8 ????45 ????46 ????47 ????48 ????49 ????50 ????51 ????13 ????52 ????53 ????54 ????55 ????56 ????57 ????58	?LSEIKGVIVHRLEGV ?GILESRGIKARITHVDTESY ?KKQYIKANSKFIGITEL ?KKFNNFTVSFWLRVPKVSASHL ?KKLRRLLYMIYMSGLAVRVHVSKEEQYYDY ?YDPNYLRTDSDKDRFLQTMVKLFNRIK ?GAYARCPNGTRALTVAELRGNAEL ?FFLLTRILTIPQSLD ?VSFGVWIRTPPAYRPPNAPIL ?SDFFPSVRDLLDTASALYRE ?PHHTALRQAILCWGELMTLA ?WVRDIIDDFTNESSQKT ?RAGRAILHIPTRIRQGLER ?AVAEGTDRVIEVLQRAGRAIL ?ALNIWDRFDVFTLGATSGYLKGNS ?TINKPKGYVGKE ?DSETADNLEKTVAALSILPGHG ?EEIVAQSIALSSLMVAQAIPLVGELVDIGFAATNFVE ?SC ?DIEKKIAKMEKASSVFNVVNS ?KWFKTNAPNGVDEKIRI ?GLQGKIADAVKAKG ?GLAAGLVGMAADAMVEDVN ?STETGNQHHYQTRVVSNANK

Table 9

The aminoacid sequence of artificial T h epi-position

The description of Th	SEQ?ID?NO:	Aminoacid sequence
The description of Th	SEQ?ID?NO:	Aminoacid sequence	1,4,9?PALINDROMIC	?6	ISEIKGVIVHKIEGI MT??RT??TRM?TM ?L???L?V
?Syn??Th??(1,2,4)	12	?KKKIITITRIITIITTID	1,4,9?PALINDROMIC	?6	ISEIKGVIVHKIEGI MT??RT??TRM?TM ?L???L?V
?Syn??Th??(1,2,4)	12	?KKKIITITRIITIITTID	IS (1,4,9 PALINDROMIC) LF simplifies	36	ISISEIKGVIVHKIEGILF ???T??RT??TR??T
IS(1,4,9??PALINDROMIC)LF	59	ISISEIKGVIVHKIEGILF ???MT?RT??TRM?TM ????L??L??V	IS (1,4,9 PALINDROMIC) LF simplifies	36	ISISEIKGVIVHKIEGILF ???T??RT??TR??T

Table 10

Other representative peptide of the present invention

The peptide code	The description of Th epi-position	Target B peptide	The aminoacid sequence of representative peptidic constructs
The peptide code	The description of Th epi-position	Target B peptide		2249??b (Seq.ID?No:60)	1,4,9PALINDROMIC?Th ????(Seq.ID?No.6)	(C) X (39-66) (C), cyclisation (Seq.ID No.5)	??ISEIKGVIVHKIEGI-GG- ??MT??RT??TRM?TM ??L???L??V ??(C)NQ?GSFLTKGPSK??LNDRADSRRS??LWDQGN?(C)
??1902??b ??(Seq.ID?No:61)	IS (1,4,9 PALINDROMIC Th simplify free) LF (Seq.ID No. 36)	(C) X (39-66) (C), cyclisation (Seq.ID No.5)	??ISISEIKGVIVHKIEGILF-GG- ????T??RT????TR??T ??(C)NQ??GSFLTKGPSK??LNDRADSRRS??LWDQGN??(C)	2249??b (Seq.ID?No:60)	1,4,9PALINDROMIC?Th ????(Seq.ID?No.6)	(C) X (39-66) (C), cyclisation (Seq.ID No.5)
??1902??b ??(Seq.ID?No:61)	IS (1,4,9 PALINDROMIC Th simplify free) LF (Seq.ID No. 36)	(C) X (39-66) (C), cyclisation (Seq.ID No.5)		??2249?f ??(Seq.ID?No.62)	??gp41??Th ₆??(N?end)(Sq.ID?No. ??50)-GG-1,4,9?PALINDROMIC?Th ????(Seq.ID?No.6)	(C) X (39-66) (C), cyclisation (Seq.ID No.5)	AVAEGTDRVIEVLQRAGRAIL-GG- ISEIKGVIVHKIEGI-GG- MT??RT????TRM??TM ?L???L??V (C)NQ?GSFLTKGPSK?LNDRADSRRS?LWDQGN?(C)

Table 11

The immunogenicity of the representative peptide of the present invention

The animal kind	Group #	Adjuvant and immunization protocol	The peptide code	The description of representative peptidic constructs	??WPI	Log on target antigen ₁₀ELISA tires	The serum titer that neutralization analysis draws (HIV-1 Clade B VL135)
									50% suppresses	90% suppresses
							Cavy	????1	50% suppresses	90% suppresses	Oil-in-water emulsion (0,3, oil-in-water emulsion 6 WPI)	????P2249?b ????(Seq?ID ????No.60)	1,4,9 PALINDROMIC Th-GG-(C) X (39-66) (C), cyclisation (SeqID No.6)-GG-(SeqID No.5)	??5	????3.806	????23	????12
??8	????4.000	????161	????56											??5	????3.806	????23	????12
??8	????4.000	????161	????56	?10	????4.500	????382			????118
?12	????5.000	????1078	????324	?10	????4.500	????382			????118
?12	????5.000	????1078	????324	????2	Oil-in-water emulsion (0,3,6 WPI)	p1902?b (Seq?ID ?No.61)			IS (1,4,9 PALINDROMIC Th simplified lib) LF-GG-(C) X (39-66) (C), cyclisation (Seq ID No.36)-GG-(Seq ID No.5)	?5				????4.684	????24	????12
?8	?????over	????178	????95							?5				????4.684	????24	????12
?8	?????over	????178	????95					?10		?????over	????345	????111
Pig	????1	Oil-in-water emulsion (0,3, oil-in-water emulsion 6 WPI)	????p1902?b ????(Seq?ID ????No.61)					?10		?????over	????345	????111	IS (1,4,9 PALINDROMIC Th simplify free) LF-GG-(C) X (39-66) (C), cyclisation (Seq ID No.36)-GG-(Seq ID No.5)	??5	????4.500	????106	????35
				??8	????5.050	?????209	????102							??5	????4.500	????106	????35
				??8	????5.050	?????209	????102	????2	Oil-in-water emulsion (0,3,6 WPI)	????p2249f ????(Seq?ID ????No.62)	????gp41?Th ₆(N end)-GG-1,4,9 PALINDROMIC Th-GG-(C) X (39-66) (C), cyclisation (Seq ID No.50)-GG-(Seq ID No.6)-GG-(Seq ID No.5)	??5		?????4.688	??????99	????31
	??8	?????5.237	?????496	????140								??5		?????4.688	??????99	????31
	??8	?????5.237	?????496	????140	Baboon	????1	Oil-in-water emulsion (0,3,6 WPI)					????p2249f ????(Seq?ID ????No.62)	Gp41 Th6 (N end)-GG-1,4,9 PALINDROMIC Th-GG-(C) X (39-66) (C), cyclisation (Seq ID No.50)-GG-(Seq ID No.6)-GG-(Seq ID No.5)	??15	?????4.570	??????25	????11
??23	?????4.894	??????37	????12											??15	?????4.570	??????25	????11
??23	?????4.894	??????37	????12	??38				?????4.689	??????45	????13

Sequence table (1) total information:

(ⅰ) applicant: associating Biomedicines, Inc., etc.

(ⅱ) denomination of invention: the peptide combinations that is used to prevent and treat HIV infection and Immunological diseases

(ⅲ) sequence number: 61

(ⅳ) address:

(A) address: MORGAN ﹠amp; FINNEGAN

(B) street: 345 Park Avenue

(C) city: New York

(D) state: NY

(E) country: USA

(F) postcode: 10154-0054

(ⅴ) computer-reader form:

(A) medium type: floppy disk

(B) computer: IBM PC compatible

(C) operating system: PC-DOS/MS-DOS

(D) software: Word 7.0

(ⅵ) request for data formerly:

(A) application number: US 09/100,409

(B) applying date: 20-JUNE-1998

(C) classification number: 514

(ⅶ) current request for data:

(A) application number: undetermined

(B) applying date: 21-JUNE-1999

(C) classification number:

(ⅷ) agency and proxy's information:

(A) title: MARIA C.H.LIN, ESQ.

(B) number of registration: 29,323

(C) reference/recording mechanism: 1151-4154PCl

(ⅸ) telecommunication information:

(A) phone: 212-758-4800

(B) information of fax: 212-751-6849 (2) SEQ ID NO:1:

(ⅰ) sequence signature:

(A) length: 433 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:1:Lys Lys Val Val Leu Gly Lys Lys Gly Asp Thr Val1 5 10Glu Leu Thr Cys Thr Ala Ser Gln Lys Lys Ser Ile

15??????????????????20Gln?Phe?His?Trp?Lys?Asn?Ser?Asn?Gln?Ile?Lys?Ile25?????????????????30???????????????????35Leu?Gly?Asn?Gln?Gly?Ser?Phe?Leu?Thr?Lys?Gly?Pro

40???????????????????45Ser?Lys?Leu?Asn?Asp?Arg?Ala?Asp?Ser?Arg?Arg?Ser???50???????????????????55??????????????????60Leu?Trp?Asp?Gln?Gly?Asn?Phe?Pro?Leu?Ile?Ile?Lys

65??????????????????70Asn?Leu?Lys?Ile?Glu?Asp?Ser?Asp?Thr?Tyr?Ile?Cys

75??????????????????80Glu?Val?Glu?Asp?Gln?Lys?Glu?Glu?Val?Gln?Leu?Leu85??????????????????90??????????????????95Val?Phe?Gly?Leu?Thr?Ala?Asn?Ser?Asp?Thr?His?Leu

100?????????????????105Leu?Gln?Gly?Gln?Ser?Leu?Thr?Leu?Thr?Leu?Glu?Ser???110??????????115?????????????????????????120Pro?Pro?Gly?Ser?Ser?Pro?Ser?Val?Gln?Cys?Arg?Ser

125?????????????????130Pro?Arg?Gly?Lys?Asn?Ile?Gln?Gly?Gly?Lys?Thr?Leu

135?????????????????140Ser?Val?Ser?Gln?Leu?Glu?Leu?Gln?Asp?Ser?Gly?Thr145?????????????????150?????????????????155Trp?Thr?Cys?Thr?Val?Leu?Gln?ASn?Gln?Lys?Lys?Val

160?????????????????165Glu?Phe?Lys?Ile?Asp?Ile?Val?Val?Leu?Ala?Phe?Gln???170??????????????????175?????????????????180Lys?Ala?Ser?Ser?Ile?Val?Tyr?Lys?Lys?Glu?Gly?Glu

185?????????????????190Gln?Val?Glu?Phe?Ser?Phe?Pro?Leu?Ala?Phe?Thr?Val

195?????????????????200Glu?Lys?Leu?Thr?Gly?Ser?Gly?Glu?Leu?Trp?Trp?Gln205?????????????????210?????????????????215Ala?Glu?Arg?Ala?Ser?Ser?Ser?Lys?Ser?Trp?Ile?Ile

220?????????????????225Phe?Asp?Leu?Lys?Asn?Lys?Glu?Val?Ser?Val?Lys?Arg

230?????????????????235?????????????????240Val?Thr?Gln?Asp?Pro?Lys?Leu?Gln?Met?Gly?Lys?Lys

245?????????????????250Leu?Pro?Leu?His?Leu?Thr?Leu?Pro?Gln?Ala?Leu?Pro

255?????????????????260Gln?Tyr?Ala?Gly?Ser?Gly?Asn?Leu?Thr?Leu?Ala?Leu265?????????????????270?????????????????275Glu?Ala?Lys?Thr?Gly?Lys?Leu?His?Gln?Glu?Val?Asn

280?????????????????285Leu?Val?Val?Met?Arg?Ala?Thr?Gln?Leu?Gln?Lys?Asn

290?????????????????295?????????????????300Leu?Thr?Cys?Glu?Val?Trp?Gly?Pro?Thr?Ser?Pro?Lys

305?????????????????310Leu?Met?Leu?Ser?Leu?Lys?Leu?Glu?Asn?Lys?Glu?Ala

315?????????????????320Lys?Val?Ser?Lys?Arg?Glu?Lys?Pro?Val?Trp?Val?Leu325?????????????????330?????????????????335Asn?Pro?Glu?Ala?Gly?Met?Trp?Gln?Cys?Leu?Leu?Ser

340?????????????????345Asp?Ser?Ser?Gln?Val?Leu?Leu?Glu?Ser?Asn?Ile?Lys

350?????????????????355?????????????????360Val?Leu?Pro?Thr?Trp?Ser?Thr?Pro?Val?Gln?Pro?Met

365?????????????????370Ala?Leu?Ile?Val?Leu?Gly?Gly?Val?Ala?Gly?Leu?Leu

375?????????????????380Leu?Phe?Ile?Gly?Leu?Gly?Ile?Phe?Phe?Cys?Val?Arg385?????????????????390??????????????????395Cys?Arg?His?Arg?Arg?Arg?Gln?Ala?Glu?Arg?Met?Ser

400?????????????????405Gln?Ile?Lys?Arg?Leu?Leu?Ser?Glu?Lys?Lys?Thr?Cys

410?????????????????415?????????????????420Gln?Cys?Pro?His?Arg?Phe?Gln?Lys?Thr?Cys?Ser?Pro

The information of 425 430Ile (2) SEQ ID NO:2:

(ⅱ) sequence signature:

(A) length: 40 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:2:His Trp Lys Asn Trp Asn Gln Ile Lys Ile Leu Gly1 5 10Asn Gln Gly Ser Phe Leu Thr Lys Gly Pro Ser Lys

15??????????????????????20Leu?Asn?Asp?Arg?Ala?Asp?Ser?Arg?Arg?Ser?Leu?Trp25??????????????????30??????????????????35Asp?Gln?Gly?Asn

The information of 40 (2) SEQ ID NO:3:

(ⅰ) sequence signature:

(A) length: 28 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:3:Asn Gln Gly Ser Phe Leu Thr Lys Gly Pro Ser Lys1 5 10Leu Asn Asp Arg Ala Asp Ser Arg Arg Ser Leu Trp

The information of 15 20Asp Gln Gly Asn25 (2) SEQ ID NO:4:

(ⅰ) sequence signature:

(A) length: 42 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:4:Cys His Trp Lys Asn Trp Asn Gln Ile Lys Ile Leu1 5 10Gly Asn Gln Gly Ser Phe Leu Thr Lys Gly Pro Ser

15??????????????????20Lys?Leu?Asn?Asp?Arg?Ala?Asp?Ser?Arg?Arg?Ser?Leu25??????????????????30??????????????????35Trp?Asp?Gln?Gly?Asn?Cys

The information of 40 (2) SEQ ID NO:5:

(ⅰ) sequence signature:

(A) length: 30 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:5:Cys Asn Gln Gly Ser Phe Leu Thr Lys Gly Pro Ser1 5 10Lys Leu Asn Asp Arg Ala Asp Ser Arg Arg Ser Leu

The information of 15 20Trp Asp Gln Gly Asn Cys25,30 (2) SEQ ID NO:6:

(ⅰ) sequence signature:

(A) length: fifteen amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 1

(D) out of Memory :/annotate=" Ile, Met or Leu "

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 2

(D) out of Memory :/annotate=" Ser or Thr "

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 4

(D) out of Memory :/annotate=" Lys or Arg "

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 5

(D) out of Memory :/annotate=" Gly or Thr-"

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 10

(D) out of Memory :/annotate=" His or Thr "

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 11

(D) out of Memory :/annotate=" Lys or Arg "

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 12

(D) out of Memory :/annotate=" Ile, Met or Leu "

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 14

(D) out of Memory :/annotate=" Gly or Thr "

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 15

(D) out of Memory :/annotate=" Ile, Met or Val "

(ⅹ ⅰ) sequence description: SEQ ID NO:6:Xaa Xaa Glu Xaa Xaa Gly Val Ile Val Xaa Xaa Xaa1 5 10Glu Xaa Xaa

The information of 15 (2) SEQ ID NO:7:

(ⅰ) sequence signature:

(A) length: 16 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:7:Thr Ala Lys Ser Lys Lys Phe Pro Ser Tyr Thr Ala1 5 10Thr Tyr Gln Phe

The information of 15 (2) SEQ ID NO:8:

(ⅰ) sequence signature:

(A) length: fifteen amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:8:Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln1 5 10Ser Leu Asp

The information of 15 (2) SEQ ID NO:9:

(ⅰ) sequence signature:

(A) length: six amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:9:Pro Pro Xaa Pro Xaa Pro1 5 (2) SEQ ID NO:1O:

(ⅰ) sequence signature:

(A) length: 46 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:10:Cys His Trp Lys Asn Trp Asn Gln Ile Lys Ile Leu1 5 10Gly Asn Gln Gly Ser Phe Leu Thr Lys Gly Pro Ser

15??????????????????20Lys?Leu?Asn?Asp?Arg?Ala?Asp?Ser?Arg?Arg?Ser?Leu25??????????????????30??????????????????35Trp?Asp?Gln?Gly?Asn?Cys?Pro?Leu?Ile?Ile

The information of 40 45 (2) SEQ ID NO:11:

(ⅰ) sequence signature:

(A) length: 34 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:11:Cys Asn Gln Gly Ser Phe Leu Thr Lys Gly Pro Ser1 5 10Lys Leu Asn Asp Arg Ala Asp Ser Arg Arg Ser Leu

The information of 15 20Trp Asp Gln Gly Asn Cys Pro Leu Ile Ile25,30 (2) SEQ ID NO:12:

(ⅰ) sequence signature:

(A) length: 18 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:12:Lys Lys Lys Ile Ile Thr Ile Thr Arg Ile Ile Thr1 5 10Ile Ile Thr Thr Ile Asp

The information of 15 (2) SEQ ID NO:13:

(ⅰ) sequence signature:

(A) length: 12 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:13:Thr Ile Asn Lys Pro Lys Gly Tyr Val Gly Lys Glu1 5 10 (2) SEQ ID NO:14:

(ⅰ) sequence signature:

(A) length: 13 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:14:Phe Leu Thr Lys Gly Pro Ser Lys Leu Asn Asp Arg Ala1 5 10 (2) SEQ ID NO:15:

(ⅰ) sequence signature:

(A) length: 12 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:15:Cys Gly Asn Gln Gly Ser Phe Leu Thr Lys Gly Cys1 5 10 (2) SEQ ID NO:16:

(ⅰ) sequence signature:

(A) length: 18 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:16:Gly Pro Ser Lys Leu Asn Asp Arg Ala Asp Ser Arg1 5 10Arg Ser Leu Trp Asp Gln

The information of 15 (2) SEQ ID NO:17:

(ⅰ) sequence signature:

(A) length: 7 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:17:Asn Gln Gly Ser Phe Leu Thr1 5 (2) SEQ ID NO:18:

(ⅰ) sequence signature:

(A) length: 12 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:18:Cys Ile Leu Gly Asn Gln Gly Ser Phe Leu Thr Cys1 5 10 (2) SEQ ID NO:19:

(ⅰ) sequence signature:

(A) length: 29 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:19:Asn Gln Gly Ser Phe Leu Thr Lys Gly Pro Ser Lys1 5 10Leu Asn Asp Arg Ala Asp Ser Arg Arg Ser Leu Trp

The information of 15 20Asp Gln Gly Asn Phe25 (2) SEQ ID NO:20:

(ⅰ) sequence signature:

(A) length: 61 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:20:Ser Lys Leu Asn Asp Arg Ala Asp Ser Arg Arg Ser1 5 10Leu Trp Asp Gln Gly Asn Phe Pro Leu Ile Ile Lys

15??????????????????20Asn?Leu?Lys?Ile?Glu?Asp?Ser?Asp?Thr?Tyr?Ile?Cys25??????????????????30??????????????????35Glu?Val?Glu?Asp?Gln?Lys?Glu?Glu?Val?Gln?Leu?Leu

40??????????????????45Val?Phe?Gly?Leu?Thr?Ala?Asn?Ser?Asp?Thr?His?Leu

The information of 50 55 60Leu (2) SEQ ID NO:21:

(ⅰ) sequence signature:

(A) length: 14 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:21:Cys Ile Leu Gly Asn Gln Gly Ser Phe Leu Thr Lys1 5 10Gly Cys (2) SEQ ID NO:22:

(ⅰ) sequence signature:

(A) length: 20 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:22:Cys Ser Phe Leu Thr Lys Gly Pro Ser Lys Leu Asn1 5 10Asp Arg Ala Asp Ser Arg Arg Cys

The information of 15 20 (2) SEQ ID NO:23:

(ⅰ) sequence signature:

(A) length: fifteen amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:23:Cys Leu Thr Lys Gly Pro Ser Lys Leu Asn Asp Arg1 5 10Ala Asp Cys

The information of 15 (2) SEQ ID NO:24:

(ⅰ) sequence signature:

(A) length: 12 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:24:Cys Lys Gly Pro Ser Lys Leu Asn Asp Arg Ala Cys1 5 10 (2) SEQ ID NO:25:

(ⅰ) sequence signature:

(A) length: 20 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:25:Cys Ser Asp Thr Tyr Ile Cys Glu Val Glu Asp Gln1 5 10Lys Glu Glu Val Gln Leu Leu Cys

The information of 15 20 (2) SEQ ID NO:26:

(ⅰ) sequence signature:

(A) length: ten amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:26:Cys Gly Asn Gln Gly Ser Phe Leu Thr Cys1 5 10 (2) SEQ ID NO:27:

(ⅰ) sequence signature:

(A) length: eight amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:27:Cys Asn Gln Gly Ser Phe Leu Cys1 5 (2) SEQ ID NO:28:

(ⅰ) sequence signature:

(A) length: six amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:28:Cys Gln Gly Ser Phe Cys1 5 (2) SEQ ID NO:29:

(ⅰ) sequence signature:

(A) length: 5 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:29:Cys Asn Thr Arg Cys1 5 (2) SEQ ID NO:30:

(ⅰ) sequence signature:

(A) length: 7 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:30:Cys Leu Asn Thr Arg Ala Cys1 5 (2) SEQ ID NO:31:

(ⅰ) sequence signature:

(A) length: 7 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:31:Cys Pro Ser Lys Leu Asn Cys1 5 (2) SEQ ID NO:32:

(ⅰ) sequence signature:

(A) length: 77 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:32:Thr Ala Lys Ser Lys Lys Phe Pro Ser Tyr Thr Ala1 5 10Thr Tyr Gln Phe Gly Gly Phe Phe Leu Leu Thr Arg

15??????????????????20Ile?Leu?Thr?Ile?Pro?Gln?Ser?Leu?Asp?Gly?Gly?Cys25??????????????????30??????????????????35His?Trp?Lys?Asn?Trp?Asn?Gln?Ile?Lys?Ile?Leu?Gly

40??????????????????45Asn?Gln?Gly?Ser?Phe?Leu?Thr?Lys?Gly?Pro?Ser?Lys

50??????????????????55??????????????????60Leu?Asn?Asp?Arg?Ala?Asp?Ser?Arg?Arg?Ser?Leu?Trp

65??????????????????70Asp?Gln?Gly?Asn?Cys

The information of 75 (2) SEQ ID NO:33:

(ⅰ) sequence signature:

(A) length: 69 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:33:Thr Ala Lys Ser Lys Lys Phe Pro Ser Tyr Thr Ala1 5 10Thr Tyr Gln Phe Gly Gly Phe Phe Leu Leu Thr Arg

15??????????????????20Ile?Leu?Thr?Ile?Pro?Gln?Ser?Leu?Asp?Gly?Gly?Cys25??????????????????30??????????????????35Asn?Gln?Gly?Ser?Phe?Leu?Thr?Lys?Gly?Pro?Ser?Lys

40??????????????????45Leu?Asn?Asp?Arg?Ala?Asp?Ser?Arg?Arg?Ser?Leu?Trp???50???????????????????55??????????????????60Asp?Gln?Gly?Asn?Cys?Pro?Leu?Ile?Ile

The information of 65 (2) SEQ ID NO:34:

(ⅰ) sequence signature:

(A) length: 81 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:34:Thr Ala Lys Ser Lys Lys Phe Pro Ser Tyr Thr Ala1 5 10Thr Tyr Gln Phe Gly Gly Phe Phe Leu Leu Thr Arg

40??????????????????45Asn?Gln?Gly?Ser?Phe?Leu?Thr?Lys?Gly?Pro?Ser?Lys

65??????????????????70Asp?Gln?Gly?Asn?Cys?Pro?Leu?Ile?Ile

The information of 75 80 (2) SEQ ID NO:35:

(ⅰ) sequence signature:

(A) length: 64 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:35:Thr Ala Lys Ser Lys Lys Phe Pro Ser Tyr Thr Ala1 5 10Thr Tyr Gln Phe Gly Gly Phe Phe Leu Leu Thr Arg

The information of 40 45Leu Asn Asp Arg Ala Asp Ser Arg Arg Ser Leu Trp, 50 55 60Asp Gln Gly Cys (2) SEQ ID NO:36:

(ⅰ) sequence signature:

(A) length: 19 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 4

(D) out of Memory :/annotate=" Ser or Thr "

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 7

(D) out of Memory :/annotate=" Lys or Arg "

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 8

(D) out of Memory :/annotate=" Gly or Thr "

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 12

(D) out of Memory :/annotate=" His or Thr "

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 13

(D) out of Memory :/notes=" Lys or Arg " be characteristic (ⅸ):

(A) title/keyword: decorating site

(B) position: 16

(D) out of Memory :/annotate=" Gly or Thr "

(ⅹ ⅰ) sequence description: SEQ ID NO:36:Ile Ser Ile Xaa Glu Ile Xaa Xaa Val Ile Val Xaa1 5 10Xaa Ile Glu Xaa Ile Leu Phe

The information of 15 (2) SEQ ID NO:37:

(ⅰ) sequence signature:

(A) length: 50 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:37:Lys Lys Lys Ile Ile Thr Ile Thr Arg Ile Ile Thr1 5 10Ile Ile Thr Thr Ile Asp Gly Gly Cys Asn Gln Gly

15??????????????????20Ser?Phe?Leu?Thr?Lys?Gly?Pro?Ser?Lys?Leu?Asn?Asp25??????????????????30??????????????????35Arg?Ala?Asp?Ser?Arg?Arg?Ser?Leu?Trp?Asp?Gln?Gly

40??????????????????45Asn?Cys

The information of 50 (2) SEQ ID NO:38:

(ⅰ) sequence signature:

(A) length: fifteen amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:38:Leu Ser Glu Ile Lys Gly Val Ile Val His Arg Leu1 5 10Glu Gly Val

The information of 15 (2) SEQ ID NO:39:

(ⅰ) sequence signature:

(A) length: 20 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:39:Gly Ile Leu Glu Ser Arg Gly Ile Lys Ala Arg Ile1 5 10Thr His Val Asp Thr Glu Ser Tyr

The information of 15 20 (2) SEQ ID NO:40:

(ⅰ) sequence signature:

(A) length: 17 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:40:Lys Lys Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile1 5 10Gly Ile Thr Glu Leu

The information of 15 (2) SEQ ID NO:41:

(ⅰ) sequence signature:

(A) length: 22 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:41:Lys Lys Phe Asn Asn Phe Thr Val Ser Phe Trp Leu1 5 10Arg Val Pro Lys Val Ser Ala Ser His Leu

The information of 15 20 (2) SEQ ID NO:42:

(ⅰ) sequence signature:

(A) length: 30 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:42:Lys Lys Leu Arg Arg Leu Leu Tyr Met Ile Tyr Met1 5 10Ser Gly Leu Ala Val Arg Val His Val Ser Lys Glu

The information of 15 20Glu Gln Tyr Tyr Asp Tyr25,30 (2) SEQ ID NO:43:

(ⅰ) sequence signature:

(A) length: 27 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:43:Tyr Asp Pro Asn Tyr Leu Arg Thr Asp Ser Asp Lys1 5 10Asp Arg Phe Leu Gln Thr Met Val Lys Leu Phe Asn

The information of 15 20Arg Ile Lys25 (2) SEQ ID NO:44:

(ⅰ) sequence signature:

(A) length: 24 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:44:Gly Ala Tyr Ala Arg Cys Pro Asn Gly Thr Arg Ala1 5 10Leu Thr Val Ala Glu Leu Arg Gly Asn Ala Glu Leu

The information of 15 20 (2) SEQ ID NO:45:

(ⅰ) sequence signature:

(A) length: 21 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:45:Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala1 5 10Tyr Arg Pro Pro Asn Ala Pro Ile Leu

The information of 15 20 (2) SEQ ID NO:46:

(ⅰ) sequence signature:

(A) length: 20 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:46:Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp1 5 10Thr Ala Ser Ala Leu Tyr Arg Glu

The information of 15 20 (2) SEQ ID NO:47:

(ⅰ) sequence signature:

(A) length: 20 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:47:Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys1 5 10Trp Gly Glu Leu Met Thr Leu Ala

The information of 15 20 (2) SEQ ID NO:48:

(ⅰ) sequence signature:

(A) length: 17 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:48:Trp Val Arg Asp Ile Ile Asp Asp Phe Thr Asn Glu1 5 10Ser Ser Gln Lys Thr

The information of 15 (2) SEQ ID NO:4 9:

(ⅰ) sequence signature:

(A) length: 19 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:49:Arg Ala Gly Arg Ala Ile Leu His Ile Pro Thr Arg1 5 10Ile Arg Gln Gly Leu Glu Arg

The information of 15 (2) SEQ ID NO:50:

(ⅰ) sequence signature:

(A) length: 21 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:50:Ala Val Ala Glu Gly Thr Asp Arg Val Ile Glu Val1 5 10Leu Gln Arg Ala Gly Arg Ala Ile Leu

The information of 15 20 (2) SEQ ID NO:51:

(ⅰ) sequence signature:

(A) length: 25 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide (ⅹ ⅰ) sequence description: SEQ ID NO:51:Ala Leu Asn Ile Trp Asp Arg Phe Asp Val Phe Ser1 5 10Thr Leu Gly Ala Thr Ser Gly Tyr Leu Lys Gly Asn

The information of 15 20Ser25 (2) SEQ ID NO:52:

(ⅰ) sequence signature:

(A) length: 22 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:52:Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Val1 5 10Ala Ala Leu Ser Ile Leu Pro Gly His Gly

15 20 (information of 2 SEQ ID NO:53:

(ⅰ) sequence signature:

(A) length: 39 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:53:Glu Glu Ile Val Ala Gln Ser Ile Ala Leu Ser Ser1 5 10Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu

The information of 15 20Leu Val Asp Ile Gly Phe Ala Ala Thr Asn Phe Val25,30 35Glu Ser Cys (2) SEQ ID NO:54:

(ⅰ) sequence signature:

(A) length: 21 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:54:Asp Ile Glu Lys Lys Ile Ala Lys Met Glu Lys Ala1 5 10Ser Ser Val Phe Asn Val Val Asn Ser

The information of 15 20 (2) SEQ ID NO:55:

(ⅰ) sequence signature:

(A) length: 17 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:55:Lys Trp Phe Lys Thr Asn Ala Pro ASn Gly Val Asp1 5 10Glu Lys Ile Arg Ile

The information of 15 (2) SEQ ID NO:56:

(ⅰ) sequence signature:

(A) length: 14 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:56:Gly Leu Gln Gly Lys Ile Ala Asp Ala Val Lys Ala1 5 10Lys Gly (2) SEQ ID NO:57:

(ⅰ) sequence signature:

(A) length: 19 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:57:Gly Leu Ala Ala Gly Leu Val Gly Met Ala Ala Asp1 5 10Ala Met Val Glu Asp Val Asn

The information of 15 (2) SEQ ID NO:58:

(ⅰ) sequence signature:

(A) length: 20 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:58:Ser Thr Glu Thr Gly Asn Gln His His Tyr Gln Thr1 5 10Arg Val Val Ser Asn Ala Asn Lys

The information of 15 20 (2) SEQ ID NO:59:

(ⅰ) sequence signature:

(A) length: 19 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 3

(D) out of Memory :/annotate=" Ile, Met or Leu "

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 4

(D) out of Memory :/annotate=" Ser or Thr "

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 7

(D) out of Memory :/annotate=" Lys or Arg "

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 8

(D) out of Memory :/annotate=" Gly or Thr "

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 12

(D) out of Memory :/annotate=" His or Thr "

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 13

(D) out of Memory :/annotate=" Lys or Arg "

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 14

(D) out of Memory :/annotate=" Ile, Met or Leu "

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 16

(D) out of Memory :/annotate=" Gly or Thr "

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 17

(D) out of Memory :/annotate=" Ile, Met or Val "

(ⅹ ⅰ) sequence description: SEQ ID NO:59:Ile Ser Xaa Xaa Glu Ile Xaa Xaa Val Ile Val Xaa1 5 10Xaa Xaa Glu Xaa Xaa Leu Phe

The information of 15 (2) SEQ ID NO:60:

(ⅰ) sequence signature:

(A) length: 51 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 4

(D) out of Memory :/annotate=" Ser or Thr "

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 7

(D) out of Memory :/annotate=" Lys or Arg "

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 8

(D) out of Memory :/annotate=" Gly or Thr "

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 12

(D) out of Memory :/annotate=" His or Thr "

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 13

(D) out of Memory :/annotate=" Lys or Arg "

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 16

(D) out of Memory :/annotate=" Gly or Thr "

(ⅹ ⅰ) sequence description: SEQ ID NO:60:Ile Ser Ile Xaa Glu Ile Xaa Xaa Val Ile Val Xaa1 5 10Xaa Ile Glu Xaa Ile Leu Phe Gly Gly Cys Asn Gln

15??????????????????20Gly?Ser?Phe?Leu?Thr?Lys?Gly?Pro?Ser?Lys?Leu?Asn25??????????????????30??????????????????35Asp?Arg?Ala?Asp?Ser?Arg?Arg?Ser?Leu?Trp?Asp?Gln

40??????????????????45Gly?Asn?Cys

The information of 50 (2) SEQ ID NO:61:

(ⅰ) sequence signature:

(A) length: 70 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 24

(D) out of Memory :/annotate=" Ile, Met or Leu "

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 25

(D) out of Memory :/annotate=" Ser or Thr "

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 27

(D) out of Memory :/annotate=" Lys or Arg "

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 28

(D) out of Memory :/annotate=" Gly or Thr "

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 33

(D) out of Memory :/annotate=" His or Thr "

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 34

(D) out of Memory :/annotate=" Lys or Arg "

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 35

(D) out of Memory :/annotate=" Ile, Met or Leu "

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 37

(D) out of Memory :/annotate=" Gly or Thr "

(ⅸ) characteristic:

(A) title/keyword: decorating site

(B) position: 38

(D) out of Memory :/annotate=" Ile, Met or Val "

(ⅹ ⅰ) sequence description: SEQ ID NO:61:Ala Val Ala Glu Gly Thr Asp Arg Val Ile Glu Val1 5 10Leu Gln Arg Ala Gly Arg Ala Ile Leu Gly Gly Xaa

15??????????????????20Xaa?Glu?Xaa?Xaa?Gly?Val?Ile?Val?Xaa?Xaa?Xaa?Glu25??????????????????30??????????????????35Xaa?Xaa?Gly?Gly?Cys?Asn?Gln?Gly?Ser?Phe?Leu?Thr

40??????????????????45Lys?Gly?Pro?Ser?Lys?Leu?Asn?Asp?Arg?Ala?Asp?Ser

50??????????????????55??????????????????60Arg?Arg?Ser?Leu?Trp?Asp?Gln?Gly?Asn?Cys

65??????????????????70

Claims

1. CD4-CDR2 antigen peptide, wherein said antigen peptide length about 30 between about 46 amino acid; Wherein said CD4-CDR2 antigen peptide contains by separated two cysteine residues of the insertion sequence of 28 to 40 amino-acid residues; And wherein said insertion sequence is one section sequential portion of sequence of 27 to 66 residue representatives of SEQ ID NO:1, or the immunologic function analogue of 27 to 66 residues of a kind of SEQ ID NO:1.

2. the CD4-CDR2 antigen peptide of claim 1, wherein said antigen peptide is selected from SEQID NO:4, SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:11 or its immunologic function analogue.

3. a length is about 50 to about 80 amino acid whose synthetic peptides, and it contains:

(a) a kind of helper cell (Th) epi-position;

(b) a kind of CD4-CDR2 antigen peptide according to claim 1; With

(c) a kind of immunostimulation invasin structural domain.

4. a length is about 50 to about 80 amino acid whose synthetic peptides, and it contains:

(a) a kind of helper cell (Th) epi-position;

(b) a kind of CD4-CDR2 antigen peptide according to claim 2; With

(c) a kind of immunostimulation invasin structural domain.

5. a peptide or peptide binding substances that contains with the covalently bound helper T cell epitope of CD4-CDR2 antigen peptide (Th) of claim 1.

6. a peptide or peptide binding substances that contains with the covalently bound helper T cell epitope of CD4-CDR2 antigen peptide (Th) of claim 2.

7. peptide that is expressed from the next or peptide binding substances:

(A) _n-(CD4-CDR2 antigen peptide)-(B) _o-(Th) _m-X

Or

(A) _n-(Th) _m-(B) _o-(CD4-CDR2 antigen peptide)-X

Wherein:

Each A is a seed amino acid independently, or a kind of general immunostimulatory sequence;

Each B be selected from amino acid ,-NHCH (X) CH ₂SCH ₂CO-,-NHCH (X) CH ₂SCH ₂CO (Lys-of ε-N) ,-NHCH (X) CH ₂The S-succinimide (Lys-of ε-N) and-NHCH (X) CH ₂S-(succinimide)-;

Each Th is a kind of aminoacid sequence independently, and this aminoacid sequence contains a kind of helper T cell epitope or a kind of immunostimulant analogue or its fragment;

The CD4-CDR2 antigen peptide is represented the sequence of a kind of antigen peptide of claim 1;

X is a kind of amino acid whose α-COOH or α-CONH ₂

N is from 0 to about 10;

M is from 1 to about 4; With

O is from 0 to about 10.

8. peptide that is expressed from the next or peptide binding substances:

(CD4-CDR2 antigen peptide)-(B) _o-(Th) _m-(A) _n-X

Or

(Th) _m-(B) _o-(CD4-CDR2 antigen peptide)-(A) _n-X

Wherein:

Each A is a seed amino acid or a kind of general immunostimulatory sequence independently;

X is a kind of amino acid whose α-COOH or α-CONH ₂

N is from 0 to about 10;

M is from 1 to about 4; With

O is from 0 to about 10.

9. peptide that is expressed from the next or peptide binding substances:

(A) _n-(CD4-CDR2 antigen peptide)-(B) _o-(Th) _m-X

Or

(A) _n-(Th) _m-(B) _o-(CD4-CDR2 antigen peptide)-X

Wherein:

The CD4-CDR2 antigen peptide is represented the sequence of a kind of antigen peptide of claim 2;

X is a kind of amino acid whose α-COOH or α-CONH ₂

N is from 0 to about 10;

M is from 1 to about 4; With

O is from 0 to about 10.

10. peptide that is expressed from the next or peptide binding substances:

(CD4-CDR2 antigen peptide)-(B) _o-(Th) _m-(A) _n-X

Or

(Th) _m-(B) _o-(CD4-CDR2 antigen peptide)-(A) _n-X

Wherein:

Each B be selected from amino acid ,-NHCH (X) CH ₂SCH ₂CO-,-NHCH (X) CH ₂SCH ₂CO (Lys-of ε-N) ,-NHCH (X) CH ₂The S-succinimide (Lys-of ε-N), or-NHCH (X) CH ₂S-(succinimide)-;

X is a kind of amino acid whose α-COOH or α-CONH ₂

N is from 0 to about 10;

M is from 1 to about 4; With

O is from 0 to about 10.

11. one kind according to claim 3-10 one of any peptide or peptide binding substances, wherein said Th has a kind of SEQ of being selected from ID NOS:6,8,12,13,36 or the aminoacid sequence of 36-59.

12. peptide or peptide binding substances according to a claim 11, wherein said Th has the aminoacid sequence of a kind of SEQ of being selected from ID NOS:6 or 8.

13. one kind according to claim 3-10 one of any peptide or peptide binding substances, wherein n is 3, (A) ₃Be (invasin structural domain)-Gly-Gly.

14. peptide or peptide binding substances according to a claim 11, wherein at least one A partly is a kind of invasin structural domain.

15. peptide or peptide binding substances according to a claim 12, wherein at least one A partly is a kind of invasin structural domain.

16. one kind according to claim 3-10 one of any peptide or peptide binding substances, wherein said CD4-CDR2 antigen peptide is selected from SEQ ID NOS:4,5,10 or 11.

17. peptide or peptide binding substances according to a claim 11, wherein said CD4-CDR2 antigen peptide is selected from SEQ ID NOS:4,5,10 or 11.

18. peptide or peptide binding substances according to a claim 12, wherein said CD4-CDR2 antigen peptide is selected from SEQ ID NOS:4,5,10 or 11.

19. one kind is selected from SEQ ID NOS:32,33,34,35 or 60 peptide.

20. a pharmaceutical composition, it contains a kind of claim 3-10 of immune significant quantity or one of 19 peptide or peptide binding substances, also contains a kind of medicine acceptable carrier.

21. according to the pharmaceutical composition of claim 20, the described immune significant quantity of wherein said peptide or peptide binding substances arrives between the every dosage of about 1mg per kilogram of body weight at about 0.5 μ g.

22. a method of inducing the antibody of anti-surface C D4 mixture in the Mammals, it comprises the pharmaceutical composition to described administration claim 20.

23. a method of inducing the antibody of anti-surface C D4 mixture in the Mammals, it comprises the pharmaceutical composition to described administration claim 21.

24. one kind is suppressed that HIV is in conjunction with the method for CD4+ cell in the Mammals, it comprises the pharmaceutical composition to described administration claim 20.

25. one kind is suppressed that HIV is in conjunction with the method for CD4+ cell in the Mammals, it comprises the pharmaceutical composition to described administration claim 21.